Introduction

Exercise is known to protect individual from metabolic syndromes, in part by increasing gene transcription and induction of several signalling pathways vital in correcting impaired metabolic pathways related with a sedentary lifestyle. The regulation of carbohydrate and lipid metabolism is very crucial in patients with insulin resistance and type 2 diabetes. Glucose transporter protein (GLUT-4) is responsible for more than 80% of whole body glucose disposal in skeletal muscle (Shulman et al. 1990). In type 2 diabetes, insulin-mediated glucose uptake, oxidation, and storage by the skeletal muscle are critically impaired (Kelley et al. 2002). GLUT4 is an insulin-responsive glucose transporter that controls glucose uptake (Charron et al. 1999; Sparling et al. 2008). GLUT4 transports glucose from blood to the cells, thereby supporting glucose homeostasis (euglycemia). It is reported that GLUT4 levels are low in diabetes mellitus (Garvey et al. 1991; Gibbs et al. 1995; Kahn et al. 1988; Sinha et al. 1991); hence, over-expression of Glut4 transcription is seen as a means to ease type 2 diabetes (Brozinick et al. 2001; Christ-Roberts et al. 2004; Oshel et al. 2000). Moreover, Glut4 gene is regulated by the myocyte enhancing (Mef2) transcription factor that binds to its cis-elements as a hetero-dimer (Mef2a/D) resulting in GLUT4 expression (Oshel et al. 2000; Thai et al. 1998). GLUT4 expression is also regulated by nuclear respiratory factor (nrf-1), a mitochondrial transcription factor, which regulates mef2a gene to regulate Glut4 (Baar et al. 2003; Ramachandran et al. 2008). Thus, owing to their essential roles in blood sugar metabolism, these genes have become targets for pharmacological therapeutics in the management and treatment of type 2 diabetes.

The study was conducted to identify optimum time point’s increase/decrease of gene expression occurring in response to exercise. Genes express at specific various times and decay at different times. The optimum time point of induction/degradation of glucose transport and lipid metabolism gene expression were specifically targeted as well as the investigation on the optimum time point increase of Nrf-1 bound Mef2a and Cpt-1 promoters.

Methodology

Animal care

Five- to 6-week-old male Wistar rats were used for this study. All animal procedures were approved by the Animal ethics committee of the University of Witwatersrand (WITS University), RSA. Rats were fed standard rat chow and water ad libitum. Room temperature was maintained at 21–24 °C with a 12-h light/dark cycle. Their welfare and weight were checked daily. The rats were familiarized to handling as well as to exercise protocol prior to the experiment to ensure that the animals were not stressed during the experiment. The temperature of water for swimming was monitored and maintained at 35 °C to ensure that the rats did not suffer from hypothermia or overheating during exercise. Following each bout of exercise, rats were gently towel-dried and allowed to rest in their cages to ensure that they experienced minimal discomfort by being wet.

Experimental design

In this study, a total number of 36 rats were used. Rats were divided into 2 groups, namely, exercise and non-exercise (control). The purpose of these 2 groups was to investigate the level of gene expression in response to exercise compared with non-exercising rats. The exercise group were then subdivided into 8 different groups, in which 4 rats formed a group. Four rats also formed the control group (Group 9). Rats were exercised according to protocol reported by Smith et al. (2007) and Terada et al. (2001). The swimming protocol used to exercise the rats is stipulated by a flow diagram in Supplementary file (Fig. 1). From days 1 to 4, rats were housed and familiarized to handling. Thereafter, from days 5 to 8, rats were familiarized to swimming protocol as follows: on day 5, rats were subjected to 2 bouts of 17 min swimming and 3 min rest. From day 9 to day 14, rats rested in their cages in order to ensure that the familiarization protocol did not affect the experiment. From days 15 to 19, the exercise group performed 5 bouts of 17 min of swimming with 3 min of rest in between bouts. The control group remained in the cages for the entire duration of the experiment. Rats in groups 1 to 8 were then anaesthetized at different time periods: 0, 2, 4, 6, 8, 10, 12, and 15 h post exercise by intraperitoneal injection of Ketamine. Gastrocnemius muscle was dissected out for analysis, snap frozen in liquid nitrogen, and stored at −80 °C freezer in cryovial until required for use.

Fig. 1
figure 1

Exercise increases Nrf-1 gene expression to optimum level at time point 6 h post exercise. Gastrocnemius muscles were extracted 0, 2, 4, 6, 8, 10, 12, and 15 h post exercise. The Nrf-1 gene expression of 6 h post exercise showed ~ 6.3 fold increase compared with the control. The P value exercise (6 h) vs control P < 0.001 (***)

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Quantitative real-time PCR

Total RNA was isolated and purified from ~ 100 mg frozen Gastrocnemius muscle using QIAzol lysis reagent (QIAGEN Sciences, USA) and RNA clean and Concentrator-25 (Inqaba Biotech, SA). Double stranded cDNA was synthesized from ~ 3 mg of total RNA using Superscript Reverse Transcriptase III (Invi-trogen, USA). Quantitative real-time PCR were set up using SensiMix SYBER Noe ROX one-step kit (Bioline, UK) and were cycled according to the Sensi Mix kit instructions in Rotor Gene e3000 (QIAGEN Sciences, USA) qPCR machine. Real-time PCR was performed in triplicate using Rotor gene-3000 Thermo cycler PCR machine; Sensi Mix SYBER green PCR reagent and primers (Integrated DNA technologies, US) were used to amplify the genes. The primers are Nrf-1 forward 5-TTACTCTGCTGTGGCTGATGG-3 reverse 5′-CCTCTGATGCTTGCGTCGTCT-3′; Mef2a forward: ′5-CCG CCT CAG AAC TTC TCA ATG-3′ reverse ′5-TTG GAG AGG CCC TTG AGT TTA C-3′. Amplification occurred in a three-step cycle, denaturation at 95 °C for 5 s, annealing at 62 °C for 10 s and extension at 72 °C for 15 s. Relative mRNA expression was normalized to Actin reference gene (Actin forward primer- ′5-GAC GAG GCC CAG AGC AAG AGA 3′ reverse primer ′5 GGG TGT TGA AGG TCT CAA ACA 3′). Expression ratio was calculated according to relative standard method.

Chromatin immunoprecipitation assay

NRF-1 is known to bind to MEF2A and CPT-1 to regulate both glucose metabolism and lipid oxidation, respectively. To assess if exercise had any effect on NRF-1 binding activities to these promoters, we performed ChIP assay. This was used to identify the optimum time point of the increase of NRF-1 bound Mef2a and Cpt-1promoter1 promoter regions after exercise compared with the control group (Table 1).

Table 1 ChIP assay primer sequence
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Approximately 100 mg of Gastrocnemius muscle from each group (Groups 1–9) was grounded with 10 mL of 1% formaldehyde in a phosphate buffered saline (PBS: pH 7.4) to cross-link protein to DNA. Cross-linking was stopped by adding 500 µL of 2.5 M glycine, which was incubated for 5 min, centrifuged for 5 min at 13,000 rpm, and washed 3 times using 5 mL chilled PBS. Supernatant was then removed and pellet sonicated with 500 µL of Lysis buffer (50 mM -Tris HCl pH 8.0, 10 mM EDTA, 10% SDS, 10 mM Na4P2O7, 20 mM NaF, 4 mM Na3VO4, 1 × RCPI) on ice. The resultant chromatin was sheared 8 times for 15 s (1 min rest in between the cycles) at a maximum intensity of 33% on ice to achieve size ~ 300 to 1000 bp. The sonicated sample was then centrifuged at 13,000 rpm for 10 min, and the supernatant was transferred to a new tube. One hundred microlitres of the resultant supernatant containing chromatin was diluted with 900 µL of ChIP dilution buffer (0.01% SDS, 1% Triton-X100, 1.2 mM EDTA, 16.7 mM Tris–HCl (pH 8) and 167 mM NaCl). Thirty microlitres of sample from each group was kept as an input sample (IN) and stored at −80 °C to cross-link together with the immune-precipitation (IP) sample. The remaining 970 µL sample from each group was used for IP.

Pre-clearing of endogenous antibodies from the IP sample was done by adding 30 µL of 50% agarose beads/Salmon sperm (Santa Cruz Biotechnology, US), followed by incubation at 4 °C on a rotating flat-form using VWR tube rotator for 2 h. The sample was then centrifuged to pellet beads at 2000 rpm for 2 min at 4 °C, and the supernatant was transferred to a new tube. Immunoprecipitation was allowed by incubating samples with 5 µL of monoclonal anti-NRF-1 (Sigma) at 4 °C (cold room) for 48 h, rotating gently. After incubation, immuno-complexes were precipitated with 40 µL of 50% slurry agarose/salmon sperm for 6 h in a rotating flat form at 4 °C. Beads were pelleted by centrifuge at 5000 rpm for 1 min at 4 °C, and pellets from the IP sample were sequentially washed with different buffers and centrifuged at 2000 rpm: (1) low salt wash buffer, 1 mL 1 × 5 min at 4 °C (0.1% SDS, 1% Triton-X100, 2 mM EDTA, 20 mM Tris–HCl (pH 8.0), 150 mM NaCl); (2) high salt buffer, 1 mL 1 × 5 min at 4 °C (0.1% SDS, 1% Triton-X100, 2 mM EDTA, 20 mM Tris–HCl (pH 8.0), 500 mM NaCl); (3) lithium chloride wash buffer, 1 mL 1 × 5 min at 4 °C (0.25 M LiCl, 1 mM EDTA, 10 mM Tris–HCl (pH 8.0); and (4) Tris EDTA buffer, 1 mL 2 × 5 min at room temperature (RT) (1 mM EDTA, 10 mM Tris–HCl). After washing, beads were suspended in a fresh Elution buffer (1% SDS, 1 M NaHCO3).

Elution was done with 150 µL elution buffer for 15 min at RT and repeated twice to make a 300-µL final volume. The above-mentioned IN sample was used from this point onwards. Ninety microlitres of elution buffer and 7.5 µL of 5 M NaCl (final concentration: 300 nM) were added to the 30 µL of IN sample mentioned above. IP and IN samples were then reverse cross-linked with 5 M NaCl (Final: 300 nM) at 65 °C overnight. This was followed by an addition of 0.5 M EDTA, 1 M Tris- HCl (pH 6.5) and proteinase K (stock: 10 µg/µL) and incubation at 45 °C for 1 h. This was to destroy the protein in the sample.

To assess the amount of NRF-1 bound to Mef2a, a 315-bp fragment Mef2a gene was PCR amplified from IN and IP samples. Quantitative real-time PCR also used to determine the amount of NRF-1 bound Mef2a and Cpt-1. Real-time PCR was performed in triplicate using Rotorgene-3000 thermo cycler PCR machine, Sensimix syber green PCR reagents (Bioline, UK), and primers were used to amplify regions of Mef2a. In brief, 2 µL of DNA were added to the following reagents: 10 µL of Sensi fast, 1 µL of 10 µM forward primer, 1 µL of 10 µM reverse primer, and made up to 20 µL with milliq water. Amplification occurred in a 3-step cycle: denaturation at 95 °C for 5 s, annealing at 55 °C for 10 s, and extension at 72 °C for 15 s. Density of DNA was assessed by running 2% agarose gel electrophoresis.

Statistical analysis

Results are presented as means ± SD. Statistical analysis was performed by one-way ANOVA followed by the Tukey’s post hoc test. The level of significance was accepted at P < 0.05. All statistical analyses were performed using GraphPad InStat 3 software. *** indicates P < 0.001, ** indicates P < 0.01, and * indicates P < 0.05.

Results and discussion

Exercise induces expression of glucose transport and lipid metabolising genes in rat skeletal muscle

NRF-1 is a transcriptional factor that regulates mitochondrial biogenesis and GLUT4 expression in parallel. This study assessed the optimum time point of Nrf-1 gene expression in response to exercise. As shown in Fig. 1, Nrf-1 gene expression of the exercise group showed significant increase compared with the control group. Nrf-1 gene expression increased to the optimum level at (~ 6.3 fold) time point at 6 h post exercise. This result indicates that the optimum time point of Nrf-1 gene expression is 6 h post exercise. As such, in intervention studies 6 h was used as the optimal time for its expression.

Furthermore, Pgc-1 gene expression in response to exercise was investigated. PGC-1 is a transcriptional factor known to co-activate NRF-1 to induce mitochondrial biogenesis and glucose transport genes. As shown in Supplementary Fig. 2, after exercise Pgc-1 gene expression showed significant increase when compared with the control group. Pgc-1 expression increased to the optimum level (~ 4.0 fold) at time point 4 h post exercise compared with other time points. This result indicates that the optimum level of Pgc-1 expression is at 4 h post exercise.

Glut4 expression was also analysed in response to exercise using qPCR. As shown in Supplementary Fig. 3, Glut4 gene expression increased after exercise as compared with the control group. After 8 h of exercise, Glut4 gene expression showed higher levels, ~ threefold increase, compared with the control. This result indicates that Glut4 gene expression increased to an optimum level at time point 15 h post exercise.

In addition, the optimum time point of Mef2a gene expression in response to exercise was investigated. Results shown in Fig. 2 indicate that gene expression of Mef2a increases after exercise as compared with the control group. After 6 h of exercise, Mef2a gene expression showed higher levels, ~ 4.2-fold increase compared with the control. This result indicates that the optimum time point of Mef2a gene expression is 6 h post exercise.

Fig. 2
figure 2

Exercise increase Mef2a gene expression to optimum level at time point 6 h post exercise. Gastrocnemius muscles were extracted 0, 2, 4, 6, 8, 10, 12, and 15 h post exercise. At 6 h post exercise, the Mef2a expression showed ~ 4.6 fold increase compared with the control group. The P value exercise (6 h) vs control P < 0.001 (***)

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Thereafter, gene expressions of Cpt-1 and Acc-1 were also assessed in response to exercise. CPT-1 is an enzyme that encoded the cpt-1 gene that controls the rate-limiting step in lipid oxidation. The result shown in Supplementary Fig. 4 indicates that Cpt-1 gene expression showed significant increase after 2, 4, 6, 8, 10, 12, and 15 h post exercise compared with the control group. Cpt-1 gene expression shows an optimum level at time point 4 h post exercise.

In addition, Acc-1 gene expression decreased after 0, 2, and 4 post exercise compared with the control (Supplementary Fig. 5). After 4 h post exercise, Acc-1 gene expression shows optimum decrease ~ 2.4 fold, decrease compared with the control. This result indicates that the optimum time point of Acc-1 gene expression is 4 h post exercise.

Exercise increases amount of NRF-1 bound Mef2a 4 h post exercise in rat skeletal muscle

NRF-1 is a transcriptional factor that regulates mitochondrial biogenesis and Glut4 expression indirectly via Mef2a. NRF-1 regulates Glut4 expression by regulating Mef2a gene expression because MEF2, which has binding sites on Glut4 promoter (Michael 2001). As such, we focussed on NRF-1 binding to its target genes, Mef2a and also Cpt-1. NRF-1 binding Mef2a promoter was assessed for how NRF-1 regulates glucose transport and its binding to Cpt-1 gene on how NRF-1 regulate fatty acid metabolism. ChIP assay was used to determine NRF-1 bound Mef2a promoter at different time intervals. The results shown in Fig. 3 indicate that exercise increases the amount of NRF-1 bound mef2a compared with the control group. Exercise increases the binding of NRF-1 to Mef2a at an optimum time point 4 h post exercise, and it shows approximately 1.8-fold increases compared the control. This indicates that the optimum time point of NRF-1 bound Mef2a is 4 h post exercise.

Fig. 3
figure 3

Exercise increases NRF-1 bound Mef2a to optimum level at time point 4 h post exercise. Gastrocnemius muscles were extracted from rats 0, 2, 4, 6, 8, 10, 12, and 15 h post exercise. The NRF-1 binding to Mef2a gene increases after exercise and it showed an optimum increase 4 h post exercise. The P value, exercise (4 h) vs control P < 0.05 (*)

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Furthermore, the amount of NRF-1 bound Cpt-1 were analysed in response to exercise. As shown in Fig. 4, NRF-1 bound Cpt-1 was changed but not statistically significant change after exercise compared with the control group.

Fig. 4
figure 4

Exercise increased NRF-1 bound Cpt-1 in rat skeletal muscle, but not statistically significant

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Conclusion

This study primarily focused on determining the optimum time point of gene expression in response to exercise. Genes are expressed at specific times and degrade very rapidly. Therefore, this study conducted to find out the optimum expression time point of genes that are responsible for glucose transport and lipid oxidation in response to exercise. This study indicates that glucose transport genes such as Nrf-1, Pgc-1, Mef2a, and Glut4 show an optimum level at time points 6, 4, 6, and 8 h post exercise, respectively. After 6 h of exercise, expression of glucose transport genes shows significant increases compared with the control. Lipid metabolism genes such as Cpt-1 show an optimum increase of gene expression 6 h post exercise, and Acc-1 shows an optimum decrease 8 h post exercise. These results indicate that most genes are highly expressed after 6 h of exercise. Furthermore, this study showed that the optimum time point increases in the level of NRF-1 bound Mef2a and Cpt-1 was 4 h and 8 h post exercise, respectively. In conclusion, the optimum time point of most glucose transport and lipid metabolism genes is 6 h post exercise.