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Int J Clin Exp Med 2016;9(2):1422-1434www.ijcem.com /ISSN:1940-5901/IJCEM0016269

Original Article Juvenile activity levels affect predisposition to metabolic syndrome induced by maternal hypoxia in male offspring rats

Jian-Hong You1,2*, Yi-Fan Zhuang3*, Jing Cheng4, Guo-Rong Lv1,4, Jing-Xian Xie5

1Department of Ultrasound, The Second Affiliated Hospital of Fujian Medical University, Quanzhou, P. R. China; 2Department of Ultrasound, Zhongshan Hospital of Xiamen University, Xiamen, P. R. China; 3Department of Gastrointestinal Surgery, Zhongshan Hospital of Xiamen University, Xiamen, P. R. China; 4Department of Clinical Medicine, Quanzhou Medical College, Quanzhou, P. R. China; 5Department of Obstetrics and Gynecology, Xiamen Maternity and Child Health Care Hospital, Xiamen, P. R. China. *Equal contributors.

Received September 16, 2015; Accepted January 21, 2016; Epub February 15, 2016; Published February 29, 2016

Abstract: Background: An adverse intrauterine environment can predispose offspring to metabolic disorders. However, fetal programming maybe reversed by postnatal lifestyle especially physical activity levels. The goal of the present study was to determine whether predisposition induced by maternal hypoxia is aggravated or reversed by juvenile activity patterns, and to study the underlying molecular mechanisms. Methods: Sprague-Dawley rats experiencing maternal hypoxia were randomly assigned to juvenile activity patterns (sedentariness, cage activity and exercise). In male offspring, body weight, adiposity index, blood-borne factors, intramuscular oxidative/anti-oxidative status and the main proteins regulating glycolipid metabolism were evaluated by biochemical assays. Results: Maternal hypoxia offspring rats were born with reduced body weight and hyperlipemia. When full grown, lipid metabolism down-regulated and lipogenesis up-regulated. Juvenile sedentariness aggravated peripheral insu-lin resistance (Lg HOMA-IR, 1.02 ± 0.1 vs. 0.83 ± 0.0, P<0.05), increased intramuscular triglyceride levels (94.63 ± 7.6 μmol/g vs. 59.08 ± 5.5 μmol/g, P<0.05), and impaired glucose tolerance. The basal expression of fatty acid binding protein 3 and carnitine palmitoyl transferase 1 decreased, while the sterol regulatory element binding tran-scription factor 1c and fatty acid synthase increased. Following insulin stimulation, changes in membrane glucose transporter 4 and Akt expression regulating glucose metabolism occurred, and juvenile exercise normalized the observed aberrations. Conclusion: Juvenile sedentariness promoted the predisposition for metabolic syndrome in maternal hypoxia offspring, which was normalized by juvenile exercise. Changes in the intramuscular glucose up-take and lipid metabolism were responsible for the metabolic reprogramming.

Keywords: Maternal hypoxia, physical activity, sedentary lifestyle, fetal programming, metabolic syndrome

Introduction

Metabolic syndrome (MS) is a cluster of condi-tions, such as increased blood pressure, high blood sugar levels, excess body fat, and abnor-mal cholesterol levels, which increase the risk of heart disease, stroke and diabetes. MS has become more widespread over the last decade, probably influenced by the prevalence of fac-tors, such as sedentary lifestyle and Western diet [1, 2]. The concept of fetal origins of adult disease [3] was introduced based on epidemio-logic and experimental evidence, indicating that adverse intrauterine environment, includ-ing hypoxia, malnutrition, caffeine etc., can pre-dispose offspring to susceptibility for metabolic

disorders, such as MS, obesity, non-alcohol fatty liver disease, and atherosclerosis [4-6]. The fetal programming may also be modified by postnatal lifestyles especially different physical activity levels [7]. Therefore, our hypothesis is that reductions in daily ambulatory activity, a sedentary lifestyle, can aggravate metabolic disorders in offspring exposed to maternal hypoxia, and that exercise training reverses the metabolic aberrations.

Maternal hypoxia is quite a common insult dur-ing pregnancy, which independent of malnutri-tion, could promote molecular markers of insu-lin resistance in adult offspring born from a hypoxic pregnancy [8]. For instance, uteropla-

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Metabolic effects of maternal hypoxia and juvenile activity

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cental insufficiency programmed glycolipid metabolism dysfunction, lowered pancreatic blood supply leading to pancreatic dysplasia and incompetence as a compensatory adapta-tion in the uterus, while generating maladapta-tion to a relative over-nutrition environment after birth [9]. Additionally, persistently advanced gluconeogenesis without suppres-sion by insulin in intrauterine growth retarda-tion (IUGR) offspring was demonstrated to con-tribute to the later impaired glucose tolerance and insulin resistance [10]. As previously reported, we have successfully established an intrauterine hypoxia model by sending preg-nant rats to a hypoxia environment in middle and late pregnancy. We demonstrated that IUGR occurred in neonates with maternal hypoxia, and early pathologic atherosclerotic changes were detected in the adult thoracic aorta [11]. Moreover, demyelination, injured axons, and damaged microvasculars were observed in the periventricular white matter in adult offspring of the same model [12]. Finally, a synergistic effect of maternal hypoxia and postnatal environment (high-fat diet) was dem-onstrated in offspring rats in the intrauterine hypoxia model contributing to vulnerability for non-alcohol fatty liver disease [13].

It is widely accepted that the pathogenesis of MS is largely attributable to physical inactivity which has been recognized by the WHO as the fourth largest risk factor for mortality causing an estimated 3.2 million deaths globally [14]. While, Lees et al. [15] pointed out that most studies on chronic metabolic diseases neglect-ed evaluating physical activity levels. Laufs et al. [16] confirmed that physical inactivity, inde-pendent of traditional risk factors, increased reactive oxygen species and inflammation, causing damages to artery endothelial function in animal experiments. Krogh-Madsen et al. [17] have shown that reduction of daily steps for 2 week significantly impaired peripheral insulin sensitivity and cardiovascular fitness in healthy, non-exercising young men. In young healthy males after bed-rest, the levels of glu-cose transporter 4 (GLUT4), hexokinase II, pro-tein kinase B/serine-threonine kinase 1 (Akt 1), and Akt 2 were found to be reduced in vastus lateralis muscle, indicating that key proteins in glucose transport, storage, and phosphoryla-tion might be responsible for sedentariness induced insulin resistance in muscle [18]. Ad- ditionally, a study demonstrated that juvenile

inactivity could initiate changes in skeletal muscle architecture and mass, and caused metabolic abnormalities contributing to late life diseases in rats [19]. Normal cage activity and sedentariness, as compared to typical behav-ioral habits, such as climbing, chasing and rus-tling during the night cycle, in adolescence seemed to be especially important [20].

Exercise bears definitive benefits for MS pre-vention and treatment compared to physical inactivity and sedentariness. In a retrospective analysis, Kodama et al. [21] found that later mortality and incidence of cardiovascular dis-eases in diabetics were closely associated with physical inactivity, but that outcome improved by movement activity regardless of type, inten-sity, or lasting time. In offspring rat with inter-generational inheritance of perinatal high fat diet-induced metabolic disorders, early post-weaning exercise could restore fatty acid meta-bolic capacity and ameliorate adverse out-comes [22]. Gatford et al. [23], demonstrated that exercise training in early life partially or completely reversed predisposition for meta-bolic disorders in offspring rats with IUGR. In IUGR offspring, whose islet β-cell function was impaired and β-cell mass was reduced due to predisposition, aerobic exercise training improved metabolic function by suppressing glucose-stimulated insulin production and decreasing hepatic glucose production [24].

Although, the association between sedentary lifestyle and chronic metabolic diseases has been emphasized based on prospective obser-vational study, animal models for the investiga-tion of underlying molecular mechanism are scarce. Moreover, differences in the effects of juvenile activity patterns on metabolism in adult offspring after maternal hypoxia have not been examined yet. Thus, the aim of this study was to establish Sprague-Dawley rat models of sedentariness, cage activity, and exercise train-ing and to determine whether the predisposi-tion to MS induced by maternal hypoxia is aggravated or reversed by different juvenile activity patterns. Moreover, the underlying molecular mechanisms were investigated.

Materials and methods

Animals

Sprague-Dawley rats (Shanghai Slack Labora- tory Animals LLC, Shanghai, China) (10 male,


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20 female, weight 170-200 g) were bred in the Laboratory Animal Center under standard con-ditions with a 12/12 hours light/dark cycle and access to standard rat chow and water ad libi-tum. All experimental procedures were in accor-dance to the National Institutes of Health guidelines, and were approved by the Animal Experimentation Ethics Committee of Quan- zhou Medical College, China.

Maternal hypoxia model

The maternal hypoxia model in rats was estab-lished according to a previously reported meth-od [11, 25]. Briefly, after two-week acclimatiza-tion, rats were mated overnight, and pregnant rats were randomly divided into an intrauterine

ing and normal posture adjustment. In the HC and HE groups, as well as the NC group, four rats were housed per litter in cages with a basal area of 1700 cm2 providing space for climbing, chasing, fighting, and other spontaneous move-ments (Figure 1A). An incremental aerobic exercise protocol as described in Gomes et al. [27] was applied with a minor modifications in the HE group. Briefly, treadmill (ZH-PT; Anhui Zheng Hua Biological Instrument Co., Ltd., Anhui, China) training was conducted during the dark cycle (8-10 p.m.), including an adap-tive stage (2 weeks) and a formal training stage (11 weeks), for three times per week. Intolerant rats were excluded based on the maximum speed at the adaptive stage. Figure 1B illus-trates the specific training protocol.

Figure 1. Postnatal activity patterns imposed on hypoxic offspring rats. A. Sedentariness (left, 300 cm2) and cage activity (right, 1700 cm2). B. Tread-mill for exercise training. C. Exercise protocol. Adaptive stage, exercise start-ed at 10 m/min for 10 minutes on a 0° incline with the speed gradually increasing 2 m/min each time by the end of the 2nd week reaching 20 m/min. Formal stage, exercise started at 10 m/min for 10 minutes on a 0° incline with the speed and duration gradually increasing 1 m/min and 5 min per week, respectively, reaching 20 m/min and 60 min.

normoxia and hypoxia group. From day 7 to 21 of pregnan-cy, the hypoxia group was housed individually inside a low oxygen concentration (10 ± 1%) plexiglas eight hours per day where the oxygen con-centration was monitored by an oxygen analyzer (S-450; IST-AIM). Normal control (NC) rats were housed in an identi-cal chamber with an oxygen concentration around 21%. After natural delivery, litter size was randomly culled to six males for 28 days nursing then weaned to ad libitum laboratory chow. The surplus neonatal rats were sacrificed for birth weight and biomark-ers detection.

Sedentary and exercise pro-tocol

Juvenile offspring rats (5 weeks old) in the maternal hypoxia model were assigned to 3 groups: sedentariness (HS), cage activity (HC), and exercise training (HE) accord-ing to methods reported by Suvorava et al. [26]. In the HS group, rats were housed indi-vidually in cages with a basal area of 300 cm2 providing space for predominantly rest-


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Intraperitoneal glucose tolerance test (IPGTT)

Upon completion of the juvenile interventions in different groups (48 h later), rats were fasted overnight and injected intraperitoneally with a 50% glucose solution (2 g/kg). Blood glucose was detected by a glucose meter on the tail (Roche Diagnostics, Basel, Switzerland) in fast-ing condition and after glucose injection (at 0, 15, 30, 60, 90, and 120 min).

Tissue harvesting and blood sampling

After overnight fasting, rats were weighted and anesthetized by intraperitoneal injection (3 ml/100 g) with 2% sodium pentobarbital. Blood samples were collected by intracardiac punc-ture (decapitation for neonates) and after cen-trifugalized at 12,000 g, 4°C for 15 min, serum specimens were stored at -20°C. Perirenal, epi-didymal, and retroperitoneal fat tissue was col-lected and weighed. Vastus lateralis muscle specimens were collected and snap frozen in

liquid nitrogen or fixed in formaldehyde. 20 mins after either vehicle or insulin (8 U/kg) administration via bolus intraperitoneal injec-tion, the same-level of vastus lateralis muscle specimens were collected and snap frozen. Specimens of neonates mixed three into one. Euthanasia was conducted by excess sodium pentobarbital intracardiac injection.

Serum biomarker detection

The concentrations of fasting glucose and blood lipids (triglycerides (TG), total cholesterol (TC), and free fatty acids (FFA)) were detected by standard enzymatic colorimetric methods with an auto-biochemical analyzer (Rapidlab- TM850, Beckman Coulter, Fullerton, CA, USA). Fasting serum insulin, insulin-like growth factor 1 (IGF-1) and interleukin-6 (IL-6) were quanti-fied by commercially available RIA kits (Beijing North Institute of Biotechnology, Beijing, China). The homeostasis model assessment (HOMA) was adopted to evaluate the insulin resistance index (IR). HOMA-IR = serum glucose (mmol/L) × insulin (mIU/L)/22.5.

Intramuscular oxidative/antioxidative state, TG, and glycogen concentration assay

All experimental procedures were in accor-dance with operational guidelines. Briefly, homogenates of vastus lateralis muscle speci-mens were prepared by ball mill grinding (Qiagen, Hilden, Germany) in normal saline or extracting solution (1:9). Total citrate synthe-tase (CS) and superoxide dismutase (SOD) activity, concentration of malondialdehyde (MDA), intramuscular TG and glycogen levels were detected by colorimetric methods with commercial kits (Njjcbio, Nanjing, China) with a microplate reader (BIO-RAD, Hercules, CA, USA) at 412 nm, 450 nm, 532 nm, 546 nm, and 620 nm, respectively.

Histopathological procedures

For light microscopic analysis, following formal-dehyde-fixation overnight, vastus lateralis mus-cle tissues were processed by standard proce-dures in gradients of alcohol and xylene, and paraffin embedded. Tissue slices of 5 μm thick-ness were hematoxylin-eosin stained, and pho-tographed with an Olympus AH-2 light micro-scope (Olympus, Japan).

Figure 2. A. Effects of maternal hypoxia on body weight in neonates. B. Effects of juvenile activity pat-terns on body weight, fat mass and adiposity index in adult offspring rats. n=10; NC, normal controls; HC, hypoxia cage activity; HS, hypoxia sedentariness; HE, hypoxia exercise; BW: body weight; FM: fat mass; Ratio: ratio of fat mass to body weight; *P<0.05, compared with NC; &P<0.05, compared with HC; #P<0.05, compared with HS.


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Western blot analysis

Western blot analysis was performed accord-ing to standard protocols. Briefly, vastus latera-lis muscle total or membrane protein was extracted using protein extraction reagent (Beyotime Institute of Biotechnology, Haimen, China) containing protease inhibitors and ph- osphatase inhibitors, and separated by elec- trophoresis on SDS-polyacrylamide gels and transferred to nitrocellulose membranes. Then muscle homogenates were incubated with a rabbit polyclonal antibody against target pro-teins (AMP-activated protein kinase (AMPK), phosphorylated AMPK (p-AMPK Thr172), fatty acid binding protein 3 (FABP3), carnitine palmi-toyl transferase 1 (CPT1), up-regulated sterol regulatory element binding transcription factor 1c (SREBP-1c), fatty acid synthase (FAS), Akt, phosphorylated Akt (p-Akt Ser473), membrane GLUT4 (m-GLUT4) and total GLUT4 (Santa Cruz Biotechnology, Santa Cruz, CA, USA. or Cell

Signaling, Beverly, MA, USA) 1:500 in skim milk at 4°C overnight. The second antibody, goat anti-rabbit IgG antibody (1:3000 in skim milk) (EarthOx, USA), was incubated for 2 h at room-temperature, and the blots were developed by the ECL method. The relative protein levels were normalized to the β-actin expression lev-els and quantified using the Image-Pro Plus (6.0) software (Media Cybernetics, Bethesda, MA, USA).

Statistical analysis

Statistical analysis was conducted using the SPSS statistical software version 17.0 (SPSS Inc., Chicago, IL, USA). Serum insulin and HOMA-IR were logarithmically transformed to normally distribution. All values were expressed as means ± SD. The analysis of variance (ANOVA) models were used to compare various treatment groups and Fisher’s paired least sig-nificant difference test was performed to deter-mine intergroup differences. Statistically sig-nificance at P<0.05 was accepted.

Results

Effect of maternal hypoxia and juvenile activ-ity patterns on general physiological index in offspring rats

Whereas maternal hypoxia in rats did not alter litter size (11.75 ± 2.1 vs. 10.34 ± 1.7 in the NC and HC group, respectively, P>0.05), the neo-nates from maternal hypoxia rats were born

Figure 3. A. Effects of maternal hypoxia on blood-borne biomarkers in neonates. B. Effects of juvenile activity patterns on blood-borne biomarkers in adult offspring rats. n=10; NC, normal controls; HC, hy-poxia cage activity; HS, hypoxia sedentariness; HE, hypoxia exercise; TG, triglyceride; FFA, free fatty acid; IGF-1, insulin-like growth factor 1; TC, cholesterol; Glu, glucose; Lg, the base 10 logarithmic transfor-mation; Ins, insulin. *P<0.05, compared with NC; &P<0.05, compared with HC; #P<0.05, compared with HS.

Figure 4. Postprandial blood glucose in adult off-spring rats during IPGTT. n=10; NC, normal controls; HC, hypoxia cage activity; HS, hypoxia sedentariness; HE, hypoxia exercise; *P<0.05, compared with NC; &P<0.05, compared with HC; #P<0.05, compared with HE.


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with lower birth weight compared to the nor-moxia control group (5.28 ± 0.48 g vs. 6.68 ± 0.41 g, P<0.05). However, the difference in body weight vanished after breastfeeding at 28 days of age (Figure 2A).

No significant change was found in fat content of adult rats after maternal hypoxia. Juvenile exercise training (HE group) resulted in a leaner phenotype in adulthood compared to the other groups (Figure 2B). No differences were found in body weight between NC, HC, and HS groups (P>0.05). Moreover fat mass was reduced in the HE group, but was increased in the HS group compared to the NC and HC groups (P<0.05). Maternal hypoxia increased the adi-posity index in the HC and HS group when com-pared to the NC group. The adiposity index was significantly elevated after sedentariness (Figure 2B, P<0.05). In contrast, the adiposity index was significantly reduced the HE group (Figure 2B, P<0.05). It is worth mentioning that explorative willingness and behavior of rats after juvenile sedentariness were reduced with obvious anxiety in the open-field assay.

Effect of maternal hypoxia and juvenile activity patterns on blood-borne biomarkers for inflam-mation, glucose and glycolipid metabolism

In neonates, maternal hypoxia resulted in a sig-nificant increase in serum lipids TG and FFA (TG, 0.4 ± 0.2 mmol/L vs. 0.2 ± 0.1 mmol/L; FFA, 314 ± 102.9 μmol/L vs. 176 ± 89.3 μmol/L; P<0.05), and IGF-1 levels while serum levels for glucose and insulin remained normal (Figure 3A).

In adult offspring rats, the above effects of maternal hypoxia on serum lipids were still

turbance (Lg HOMA-IR, 0.74 ± 0.0 vs. 0.83 ± 0.0, P<0.05).

Interestingly, circulating IL-6, an inflammatory factor, was found unchanged after maternal hypoxia (98.3 ± 23.0 ng/L vs. 90.1 ± 20.5 ng/L, P>0.05), while increased in the HE group when compared with the HS group (152.4 ± 20.4 ng/L vs. 123.2 ± 26.9 ng/L, P<0.05).

Postprandial blood glucose in IPGTT

Maternal hypoxia interacted with the juvenile sedentary lifestyle contributing to the increase of postprandial blood glucose at 15 min, 30 min, and 60 min following injection in adult off-spring rats (Figure 4). Even after 120 min, blood glucose levels were still significantly higher in the HS group compared with the NC group. In contrast, juvenile exercise down-regu-lated postprandial blood glucose levels in the adult offspring but without reaching signifi-cance comparing with HC levels.

Intramuscular oxidative/antioxidative state, TG, and glycogen concentration

Maternal hypoxia had no effect on the intra-muscular oxidative/antioxidative status in adult rats as measured by the MDA content and SOD activity (Table 1). Citrate synthetase (CS) and glycogen concentrations were found to be slightly but not significantly down-regulated after sedentariness, but were both significantly up-regulated after training (P>0.05). Thus, the changes in glycogen concentration mimicked CS activity. Juvenile sedentariness (HS) after maternal hypoxia led to the increase of the intramuscular TG concentration compared with cage activity, while exercise normalized TG sig-nificantly (P<0.05, Table 1).

Table 1. Intramuscular oxidative/antioxidative status, glycogen and triglyceride concentration in adult offspring rats (n=10)

NCHypoxia

HC HS HECS (U/mg) 162.3 ± 19.7 151.0 ± 42.1 126.1 ± 33.6 171.1 ± 37.5#

MDA (nmol/mg) 1.3 ± 0.3 1.5 ± 0.6 1.7 ± 0.4 1.5 ± 0.6SOD (U/mg) 33.8 ± 8.3 31.6 ± 7.5 30.1 ± 6.7 38.0 ± 9.9Glycogen (mg/g) 0. 9 ± 0.2 1.0 ± 0.3 0.8 ± 0.2 1.2 ± 0.3#

TG (μmol/g) 43.3 ± 5.1 59.1 ± 5.5* 94.6 ± 7.6*,& 37.2 ± 4.7&,#

NC, normal controls; HC, hypoxia cage activity; HS, hypoxia sedentariness; HE, hypoxia exercise; CS, citrate synthase; MDA, malondialdehyde; SOD, super oxide dismutase; TG, triglyceride. *P<0.05, compared with NC; &P<0.05, compared with HC; #P<0.05, compared with HS.

present (Figure 3B) with a ten-dency of increased IR (Lg HOMA-IR, 0.83 ± 0.0 vs. 0.79 ± 0.0, P>0.05). Although most blood-borne metabolic para- meters affected by maternal hypoxia were not additionally altered by postnatal seden-tariness, impaired insulin function was aggravated (Lg HOMA-IR, 1.02 ± 0.1 vs. 0.83 ± 0.0, P<0.05). Notably, exer-cise training significantly nor-malized metabolic parame-ters and insulin function dis-


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Figure 5. Western blot analysis of signal molecules regulating glycolipid metabolism in skeletal muscle of adult rats. n=10; NC, normal controls; HC, hypoxia cage activity; HS, hypoxia sedentariness; HE, hypoxia exercise; AMPK, AMP-activated protein kinase; p-AMPK, phosphorylated AMPK; FABP3, fatty acid binding protein 3; CPT1, carnitine pal-mitoyl transferase 1; SREBP-1c, sterol regulatory element binding transcription factor 1c; FAS, fatty acid synthase; Akt, serine-threonine kinase; p-Akt, phosphorylated Akt; m-GLUT4, membrane glucose transporter 4; INS-, baseline; INS+, insulin stimulation; *P<0.05, compared with NC; &P<0.05, compared with HC; #P<0.05, compared with HE; ++P<0.05, INS+ vs. INS- of the same group.


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Morphology observations after HE staining and western blot analysis of the vastus latera-lis muscle to detect specific markers for glyco-lipid metabolism

Maternal hypoxia and juvenile activity patterns did not alter structure and morphology of the vastus lateralis muscle in adult offspring rats as observed under light microscopy (data not shown).

At the molecular level in the basal state, maternal hypoxia resulted in down-regulation of p-AMPK/AMPK, FABP3, and CPT1, as well as up-regulation of SREBP1c and FAS in skeletal muscle of adult rats compared to those in NC group (P<0.05, Figure 5A, 5B). The aberrations were exacerbated in the HS group, while dra-matically alleviated after exercise. Moreover, at the base level there were no significant differ-ences in m-GLUT4/total GLUT4 and p-Akt/Akt levels among all groups compared to the NC group (Figure 5C, 5D).

In response to insulin, p-Akt/Akt levels increased significantly in all groups (P<0.05). In the HC and HS groups, insulin stimulation of p-Akt/Akt was lower compared with controls, and juvenile training normalized the ratio. In con-trast to NC, with insulin stimulation, m-GLUT4/total GLUT4 did not correspondingly increase with p-Akt/Akt in HC and HS (Figure 5C, 5D). Nevertheless, juvenile exercise strongly enhanced insulin stimulated m-GLUT4/total GLUT4 (P<0.05, Figure 5D).

Discussion

This study determined whether predisposition to MS induced by maternal hypoxia was aggra-vated or reversed by juvenile activity patterns, such as sedentariness, cage activity and exer-cise, and evaluated underlying molecular mechanisms. The main findings of our study were as follows: 1. Maternal hypoxia indepen-dently resulted in IUGR and hyperlipemia in neonates. 2. Juvenile sedentariness exacerbat-ed the susceptibility to impaired glucose toler-ance, ectopic lipid deposited and peripheral IR. 3. Metabolic aberrations were partly reversed by exercise. 4. Changes in intramuscular glu-cose up-take and lipid metabolism were responsible for the reprogramming effect of dif-ferent juvenile activity patterns in maternal hypoxia offspring.

Importantly, we established the sedentary life-style rat model by singularizing and reducing room for locomotor activity, which differed from other studies. In most experimental models, sedentary lifestyle or physical inactivity are defined by the paradigms of bed-rest, hind limb unloading, transgenic inactive mice, and detraining [17, 19]. However, bed rest is more typical for patients after surgery or paralysis, and hindlimb unloading is the preferable local braking model for patients with bone fractures. These models, together with transgenic ani-mals, are not appropriately reflecting the typi-cal changes of a sedentary lifestyle [14]. Although, detraining reflects a realistic condi-tion of low energy consumption, the persis-tence of a high basal metabolic rate would affect resting metabolism [17, 28]. The Sedentary Behavior Research Network defines “sedentary behavior” as any waking behavior, such as sitting/reclining posture, etc., which is characterized by an energy expenditure ≤1.5 metabolic equivalents [29]. We assumed that a modern sedentary lifestyle is characterized by reduced ambulatory activity, somewhat solitary but non mobility-restricted living conditions. Accordingly, our sedentariness model reflecting these conditions appears to be more suitable for investigating the effects of a sedentary life-style on average persons. It needs to be point-ed out that psychological factors induced by a solitary lifestyle are reported to affect the immune system [30]. However, no consensus on how to assess solitary and its relationship with metabolic factors in animals has been reported so far.

Our findings are supported by previous studies [8, 11, 13, 31, 32] showing that maternal hypoxia resulted in IUGR and hyperlipemia in neonates followed by a “catch-up growth” in early adulthood. Maternal hypoxia did not alter body weight, but the adipose index increased overtly in adulthood. These results were con-firmed by finding reported by Rueda-Clausen et al. [4] in a late pregnancy hypoxia model. Biron-Shental et al. [33] showed that lipid accumula-tion in trophoblast markedly increased and lipid availability was regulated after fetal hypoxia. Given that stressors, including hypoxia or low energy conditions, can trigger glycolysis and, thus, reduced glucose level can stimulate com-pensatory increases of lipolytic hormones, e.g. cortisol, both in pregnant and fetal sheep [34].


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Hence, a high level catabolic switch could cre-ate a concentration gradient driving lipid flux to the fetus. Additionally, in our model of maternal hypoxia lasted from mid to late trimester of pregnancy, which might result in an early com-ing of lipid flux to the fetus [35]. This would give rise to mass lipid metathesis deposition in fetal organs and accordingly predispose the fetus to lipotoxicity. With this in mind, we reasoned it may explain the increased levels of fetal serum FFA and TG.

Although some studies found that physical inactivity led to a stronger inflammatory reac-tion and oxidative stress in the skeletal muscle [36]. In the present study we found that serum IL-6 levels and the intramuscular oxidative bal-ance were kept unchanged after sedentari-ness. Similarly, others have reported that there was no change in pro-inflammatory factors, such as TNF-α and IL-6, between detrained and control groups [17]. Additionally, a recent study suggested that exercise induced IL-6 release which resulted in favorable regulatory effects on local fatty acid oxidation [37]. This effect may explain to a certain extent the relationship between exercise-induced serum IL-6 increas-ing and partly reversed metabolic disorders in our study.

In contrast to the observed complete reversal effect of juvenile exercise on β-cell mass in rats with IUGR [38], our data suggested that moder-ate prolonged exercise during adolescence enhanced mitochondrial function without changing muscular structure and morphology. In support of our findings, Huber et al. [39] demonstrated that long term moderate to intense exercise enhanced substrate metabo-lism in skeletal muscle without retailing the size and distribution. Interestingly, normal lactation-al feeding was shown to restore cardiomyo-cytes numbers after uteroplacental insufficien-cy [40]. According to this study, normal lacta-tional feeding would have already normalized muscular structure and morphology. In addi-tion, in accordance with the “thrifty phenotype” hypothesis [41], we discovered that the ratio of p-AMPK to total AMPK, as an energy sensor, was down-regulated in response to the postna-tal over-nutrition status relative to prenatal intrauterine hypoxia. The thrifty phenotype hypothesis proposes that the epidemiological associations between poor fetal and infant

growth and the subsequent development of type 2 diabetes and the metabolic syndrome result from the effects of poor nutrition in early life, which produces permanent changes in glu-cose-insulin metabolism. One of the expected metabolic consequences of sedentariness would be ectopic lipid deposits and blunt insu-lin sensitivity, as revealed in our study, and which was normalized again by juvenile exer-cise. Similarly, Laye et al. [42] found that inac-tivity contributed to increased fat mass inde-pendent of excessive caloric intake. Mass lipid metathesis deposits drive IRS-1 serine phos-phorylation, while insulin-stimulated activation of Akt and downstream signal signaling was diminished [43]. Increased intramuscular TG, especially accompanied by a reduction of oxi-dation ability, contributed to impaired glucose utilization and IR [44]. Additionally, exercise was proven to increase insulin sensitivity through reducing TG levels and increasing CS and β-HAD activity in patients with type 2 dia-betes [45].

Biensø et al. [18], reported that GLUT4 and gly-cogen synthase were key players responsible for 7 days bed-rest induced IR in healthy sub-jects. Also, Caponi et al. [46] showed that aero-bic exercise increased GLUT4-related glucose intake. In line with these results, we found that insulin stimulated the increase of glucose uptake (m-GLUT4/t-GLUT4 ratio), which was diminished by juvenile sedentariness in adult offspring with maternal hypoxia without corre-sponding alterations in insulin action (p-Akt/Akt ratio). Furthermore, the observed increase of p-Akt and m-GLUT4 levels were in accor-dance with an exercise improved metabolic phenotype. These findings are in accordance with these data demonstrating that sedentari-ness facilitated the occurrence of exogenous IR via perturbating insulin responsive GLUT4 translocation independent of Akt suppression in adult offspring with maternal hypoxia [47].

Meanwhile, our data showed that glycogen lev-els increased in accordance with high CS activ-ity, a marker of mitochondrial respiratory chain activity, and an increased ratio of m-GLUT4 to total GLUT4 in male rats of HE group, which indicated the increased switch of fuel utiliza-tion to glucose. Thus, increasing glucose utiliza-tion contributed to prevent and improve meta-bolic aberrations induced by maternal hypoxia.


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However, Garg et al. [24] found that exercise did not alter insulin responsive GLUT4 in skeletal muscle in malnutrition-induced IUGR female rats. Since estradiol was demonstrated to favor GLUT4 translocation in cardiac muscles [48], we are not sure whether the exercise related improvement in insulin action was related to gender differences. It was speculated that, the pre-existing increase of basal m-GLUT4 levels before exercise as a compensatory adaptation in IUGR females might partially cover exercise-dependent GLUT4 translocation.

In the present study, TG increases were associ-ated with the expression of enzymes (SREBP1c, FAS involved in de novo lipogenesis), and nega-tively correlated with enzymes (FABP3) for lipid transportation and oxidative lipid metabolism (CPT1) in sedentary adults. The experimental results implied that sedentary lifestyle or exer-cise aggravated or alleviated TG deposits via regulation of lipogenesis, lipid transportation, and β-oxidation. Similarly, the decline of FABP3 expression, a membrane related fatty acid binding protein, was an extremely important cause in hind limb unloading induced IR in humans as revealed by gene expression micro-arrays [49]. In in vitro muscle cells cultures, FABP3 was reported as a stimulator that acti-vates AS160 phosphorylation thereby enhanc-ing glucose uptake by facilitating GLUT4 trans-location [50]. Additionally, FABP3 was increased by endurance training as an adaptation to the improvement of lipid oxidation and insulin sen-sitivity [51]. On the contrary, in rats with low birth weight, Laker et al. [44] speculated exer-cise did not reprogram skeletal muscle mito-chondrial biogenesis by detecting PGC1α, a mitochondrial biogenesis markers in gastroc-nemius muscle. However, since substrate metabolism likely differs in different types of mus-cular fibers, detecting PGC1α in fast-twitch fibers (glycolysis oriented) might not fully reflect the metabolic changes induced by exercise. In addition, Huber et al. [39] introduced malnutri-tion-induced IUGR models and documented that glycogen storage in fast-twitch gastrocne-mius muscle fibers increased in association with plasma lactate accumulation after exer-cise. They speculated that exercise could increase anaerobic glucose metabolism rather than fat utilization in IUGR offspring [39]. However, the diet-induced susceptibility in their study somewhat differed from the predisposi-

tion for metabolic disorders induced by mater-nal hypoxia in our study [8]. Moreover, the dis-parity in exercise-induced glucose metabolism depended on the predisposition for metabolic disorders, as well as exercise speed, intensity, and lasting time [24]. Therefore, more research-es were needed to confirm and improve our findings.

In the present study, preliminary trials were made to introduce a new model of juvenile sed-entariness in maternal hypoxia offspring and study the effect of juvenile activity on MS pre-disposition. Our findings have important impli-cations for study design and interpretation in regard to social habits activity patterns for the investigation of metabolic aberrations in rats. Furthermore, we confirmed the juvenile period as a critical window for interventions that could prevent or aggravate maternal hypoxia related MS diseases by different activity patterns. The predisposition for MS in maternal hypoxia off-spring was exacerbated by sedentary, while regular exercise launched in early adolescence effectively reversed MS. These results confirm the findings by Laker et al. [37], suggesting that long-lasting effects of exercise are less effec-tive and lasting in adult compared to that in the adolescent stage. Given that intrauterine hypoxia of varying degrees is a very common complication in obstetrics, we recommend that regular exercise should be initiated in early life, regardless of clear experience of intrauterine hypoxia.

Nevertheless, there were several limitations of the present study. Although, IUGR induced by maternal hypoxia was widely applied as an ani-mal model in previous studies [11-13, 26], the possibility that repetitive ischemia-reperfusion may impact fetal development in a variety of different ways should be factored in. Moreover, reductions in litter size might independently impose mammary dysfunction on offspring growth as a postnatal restriction [52]. Last but not least, additional evaluation of the relation-ship between psychological stressors induced by a singularized sedentary lifestyle and glyco-lipid metabolism needs to be considered. Consequently, future studies are needed to address two major questions: 1. How to evalu-ate the effect of psychological factors during singularized residence on metabolism. 2. What is the most effective juvenile locomotor therapy


1432 Int J Clin Exp Med 2016;9(2):1422-1434

for preventing adult metabolic diseases induced by maternal hypoxia?

Conclusions

In conclusion, the sedentariness model estab-lished in our study is well suitable for investiga-tions of the effect of social habit deficiency or different lifestyles on physiology and psycholo-gy of subjects. The predisposition for MS induced by maternal hypoxia was successfully reprogrammed by different juvenile activity pat-terns. Changes in intramuscular glucose up-take, and lipid metabolism were underlying molecular mechanism of reprogramming. Futu- re studies are required to confirm our finding and to determine the most effective juvenile locomotor therapy for prevention of “fetal ori-gins of adult diseases”, especially induced by maternal hypoxia.

Acknowledgements

This research was supported by the Natural Science Foundation of China in 2012 (No. 81271587) and Natural Science Foundation of Fujian Province in 2015 (No. 2015J01363 and No. 2015J01530). We thank Medjaden Biosci- ence Limited for assisting in the preparation of this manuscript.

Disclosure of conflict of interest

None.

Address correspondence to: Guo-Rong Lv, Depar- tment of Ultrasound, The Second Affiliated Hospital of Fujian Medical University, Zhongshan North Road 34, Quanzhou 362000, P. R. China; Department of Clinical Medicine, Quanzhou Medical College, Quanzhou, P. R. China. Tel: +86 18965525702; Fax: +86 595 22791142; E-mail: [email protected]

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