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Growth hormone potentiates thyroid hormone effects on post-exercise phosphocreatine recovery in skeletal muscle
Growth Hormone & IGF Research 22 (2012) 240–244
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Growth hormone potentiates thyroid hormone effects on post-exercise phosphocreatine recovery in skeletal muscle☆ P. Kaminsky a,⁎, P.M. Walker b, J. Deibener a, F. Barbe c, E. Jeannesson c, J.M. Escanye d, B. Dousset e, M. Klein f a
Pôle des Spécialités Médicales, Service de Médecine Interne, Centre Hospitalier Universitaire de Nancy, Hôpitaux de Brabois, 54500 Vandoeuvre, France Laboratoire de Biophysique, Faculté de Médecine-Pharmacie, Université de Bourgogne, 21000 Dijon, France Laboratoire de Biochimie des Protéines, Centre Hospitalier Universitaire de Nancy, Hôpitaux de Brabois, 54500 Vandoeuvre, France d Pôle Imagerie, Centre Hospitalier Universitaire de Nancy, Hôpitaux de Brabois, 54500 Vandoeuvre, France e Laboratoire de Biochimie, Centre Hospitalier Universitaire de Nancy, Hôpital Central, 54000 Nancy, France f Service d ' Endocrinologie, Centre Hospitalier Universitaire de Nancy, Hôpitaux de Brabois, 54500 Vandoeuvre, France b c
a r t i c l e
i n f o
Article history: Received 12 July 2011 Received in revised form 31 July 2012 Accepted 2 August 2012 Available online 28 August 2012 Keywords: Nuclear magnetic resonance Skeletal muscle Mitochondrion Somatotropin Thyroid hormones Rat
a b s t r a c t Objective: The aim of the study was to determine the respective impact of thyroxine and growth hormone on in vivo skeletal mitochondrial function assessed via post exercise phosphocreatine recovery. Design: The hind leg muscles of 32 hypophysectomized rats were investigated using 31P nuclear magnetic resonance spectroscopy at rest and during the recovery period following a non tetanic stimulation of the sciatic nerve. Each rat was supplemented with hydrocortisone and was randomly assigned to one of the 4 groups: the group Hx was maintained in hypopituitarism., the group HxT was treated with 1 μg/100 g/day of thyroxine (T4), the group HxG with 0.2 IU/kg/day of recombinant human GH (rGH) and the group HxGT by both thyroxine and rGH. Inorganic phosphate (Pi), phosphocreatine (PCr) and ATP were directly measured on the spectra, permitting the calculation of the phosphorylation potential (PP). Results: At rest, the rats treated with rGH or T4 exhibited higher PCr levels than rats Hx. The recovery rates of PCr and PP were higher in rats treated with T4 than in T4-deprivated rats, suggesting improved mitochondrial function. The rats treated by both T4 and rGH showed higher PCr and PP recovery than those maintained in hypopituitarism or treated with T4 or rGH alone. Conclusions: The study demonstrates that in contrast to T4, GH given alone in hypophysectomized rats does not improve in vivo mitochondrial oxidative metabolism. Growth hormone potentiates T4 effects on oxidative metabolism. © 2012 Elsevier Ltd. All rights reserved.
1. Introduction Growth hormone (GH) and thyroid hormones (TH) play an important role in cell growth and differentiation, and are important regulators of fuel metabolism in ATP-requiring processes. Mitochondria are the primary site of skeletal energetic metabolism and ATP production. At physiological levels, TH regulates numerous genes in skeletal muscles, especially those involved in energetic metabolism [1]. Not only does TH control the in vivo expression of a selected set of nuclear genes encoding mitochondrial inner membrane proteins [2], but they are also involved in mitochondrial DNA expression [3] encoding protein complex subunits in the respiratory chain.
☆ Funds: this work was supported by a grant of the Association Française contre les Myopathies. ⁎ Corresponding author at: Pôle des Spécialités Médicales, Service de Médecine Interne orientée vers les Maladies Orphelines et Systémiques, Centre Hospitalier Universitaire de Nancy, Hôpitaux de Brabois, rue du Morvan, 54500 Vandoeuvre-lès-Nancy, France. Tel.: +33 3 83 15 40 60; fax: +33 3 83 15 79 41. E-mail address: [email protected] (P. Kaminsky). 1096-6374/$ – see front matter © 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.ghir.2012.08.001
Moreover, the presence of both GH receptors [4] and IGF-I receptors [5] has been demonstrated in skeletal muscle. Growth hormone is known to act on protein anabolism, on the promotion of lipolysis, and on the resistance to insulin-induced glucose metabolism [6–8]. In animals, GH treatment does not modify mitochondrial respiratory rate in muscle or heart [9–11]. Furthermore, GH treatment was reported to reduce expression of several enzymes regulating lipid oxidation and energy production in hypopituitary men [12]. In contrast, an increased expression of a number of nuclear or mitochondrial genes involved in mitochondrial biogenesis was induced by GH [13–15]. In addition, in vivo post-exercise phosphocreatine (PCr) recovery rate, which is directly linked to mitochondrial function [16], has been reported recently to be correlated with IGF-1 and GH levels in healthy volunteers receiving GH stimulation with GH releasing hormone-arginine [17]. Such a discrepancy may be in part explained by functional metabolic changes induced in vivo by hormonal treatment. For instance, in muscle, the hypothyroid state induces a decreased activity of glycolysis in vitro, although in vivo studies have demonstrated an increased recruitment of this pathway, with enhanced lactate and H+ production due to decreased mitochondrial function [18,19]. In addition, hormonal status
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could be different in hypopituitary animals or patients among the different studies and little is known about the interaction between TH and GH at mitochondrial level. Using 31P magnetic resonance spectroscopy ( 31P.MRS), this study was aimed at evaluating the respective effects of GH and of TH on the in vivo mitochondrial function in hypophysectomized rats. 2. Material and methods 2.1. Animal care Male Sprague–Dawley adolescent rats (initial weight of approximatively 250 g) were purchased from Charles River France, housed four per cage in a temperature- and light-controlled room (22 °C; dark cycle 12 h–12 h), and provided with drinking water and standardized food ad libitum. They were hypophysectomized by the parapharyngeal method, and maintained in hypopituitarism for 3 weeks. All rats were treated with hydrocortisone (hydrocortisone, Roussel Laboratory, France, 62.5 μg/100 g twice daily) [20]. They were randomly assigned to four experimental groups of 8 rats: - the first group (labelled Hx) was maintained in hypopituitarism; - the second group (labelled HxT) was treated with substitutive doses of thyroxine (T4 : 1 μg/100 g/day) (L-Thyroxine¨, Laboratory Roche) [21]; - the third group (labelled HxG) was treated with 0.2 IU/kg/day of recombinant human GH (rGH : Genotonorm ¨, Laboratory Pfizer) [22]; - the fourth group (labelled HxGT) was treated by both thyroxine and rGH. Hydrocortisone and growth hormone were dissolved in saline and subcutaneously injected twice daily. Thyroxine was dissolved in saline and subcutaneously injected daily. All experiments were performed subsequent to an eleven-day hormonal treatment. Rats treated with rGH were all investigated 17 h ± 2 h after the last injection. Animal experiments were conformed to guidelines on animal care and use currently applied in our country. 2.2. Stimulation protocol The details of the stimulation and 31P.MRS protocol have previously been described elsewhere [23]. Briefly, animals were anaesthetized with ketamine (200 mg/kg, ip), with additional doses (50 mg/kg) given as needed to maintain deep anaesthesia during investigation. After exposing the right sciatic nerve, an insulated platinum bipolar electrode was placed around the exposed nerve. The animal was placed in the ventral position and the head of the gastrocnemius was positioned at the geometrical centre of the receiver coil (see below). The tendons of the muscles of the posterior compartment were then passively loaded with a weight of 100 g. The nerve stimulation (3 Hz) was achieved with rectangular supramaximal pulses (15 V, 1 ms duration), resulting in isometrical non-tetanic stimulation of the entire gastrocnemius–plantaris–soleus group. Muscle stimulation was maintained for 6 min, and immediately followed by the recording of a 10 min recovery period. This frequency level was chosen in order to obtain similar PCr depletion and intracellular pH in all the experimental groups by the end of stimulation.
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1994). Shimming of the static magnetic field was performed before each experiment using the 1H Free Induction Decay (FID). Forty eight FIDs were accumulated over 1 min with a repetition time (TR) of 1.25 s and over a bandwidth of 2000 Hz. The radiofrequency pulse length was 25 μs, and this corresponded to a maximum of signal for the 1.25 s TR. The resting spectra were obtained in 5 min in 2-koctet blocks of 240 FIDs. During the muscle stimulation and the post-stimulation recovery, the spectra were acquired continuously in 1-k octet blocks of 48 FIDs. Six spectra were hence recorded during the muscle stimulation and ten spectra during the recovery period with a time resolution of 60s/spectrum. The bone signal was filtered out by a 300 Hz differential convolution. Spectra were then apodized with 10 Hz of line broadening, Fourier transformed and phase-corrected, if required. After a second-order base-line correction, the peak area of PCr, phosphomonoester (PME), inorganic phosphate (Pi) and ATP were calculated by computed integration and corrected with the saturation factors obtained as described elsewhere [23]. 2.4. Biochemical assays After acquiring the NMR data, a muscle sample from the gastrocnemius was frozen in vivo, promptly dissected, immediately immersed in liquid nitrogen and stored at −80 °C. The levels of ATP and total creatine were assessed using reversed-phase high-performance liquid chromatography (RP-HPLC). The rat was then killed by exsanguination (aortic function), the blood sample being retained for control of hormonal assays (T3 and T4: Amersham International, London, UK; IGF-I: INCSTAR Co, Minnesota, USA). 2.5. Calculations The saddle coil collected the signal from all the hind-leg muscles below the knee. Thus, only the averaged absolute values in the hindleg were assessed. At rest, the ratios of PME/ATP, Pi/ATP, and PCr/ATP were calculated from the spectra using the ATPβ peak as reference for ATP level. The absolute resting concentrations were then estimated by multiplying these ratios by the value of ATP measured via RP-HPLC. During the muscle stimulation and recovery, the peak areas of phosphate metabolites were compared with that of a sample of diphosphonate (DP) strapped to the muscles within the saddle coil [23]. The time-course of PCr was directly calculated on the spectra through the ratio PCr/DP. We also estimated the phosphorylation potential (PP), defined as: PP ¼ ½ATP=ð½ADP ½PiÞ: It was calculated using the formula: PP ¼ ½PCr=½Pi ð½H þ KckÞ=ð½totalcreatine ½PCrÞ in which ATP and total creatine were considered as constant and measured in resting muscle using RP-HPLC, and the equilibrium constant Kck as equal to 1.66 10 9 M −1 [24]. The PCr recovery was modelized by a monoexponential fit, [PCr = A + B exp (− t / T)], the recovery rate being estimated using the constant exponential term T. The PP recovery was modelized using a linear regression during the first 3 min after the end of stimulation.
2.3. NMR protocol
2.6. Statistical analysis
The 31P.MRS experiments were performed with a 2.35 T, 40 cm diameter, horizontal superconducting magnet (Brüker BNT 100, Karlsruhe, Germany), using a saddle coil (18 mm in diameter×22 mm in length), which could be simultaneously tuned to the frequencies of both 1H (100.183 MHz) and 31P (40.5545 MHz) (Klein, Kaminsky et al.
All the results were obtained from 8 rats in each experimental group and are presented as group mean ± 1 S.D. in tables or text. In order to clarify the figures, the results are however shown as mean ± S.E., as specified in the legends. One-way analysis of variance was used to indicate an overall statistical significance among the
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means of the four experimental groups. Two-way analysis of variance (model of two factors with replication) was used to compare the time-evolution of the parameters during stimulation and recovery. The comparison of multiple linear regressions was tested using covariance analysis. Two-by-two comparisons were tested using the test of Tukey for multiple comparisons of means. A p-value of less than 0.05 was considered as significant. Statistical analyses were performed using the XLSTAT2009 statistical software package (Addinsoft). 3. Results 3.1. Results observed at rest Hormonal treatment resulted in a significant rise in the levels of thyroid hormone and IGF-I at the time of 31P.MRS investigation (Table 1). Only rats treated with T4 grew during the hormonal treatment. Table 2 shows the results obtained at rest in the four experimental groups. The concentration of ATP was significantly higher in all rats treated with T4 (groups HxT, and HxGT) than in T4-deprivated rats (Hx and HxG), while the levels of total creatine did not differ among the four groups. For 31P.MRS data, no differences were found for the Pi levels. However, PCr was lower in rats maintained in hypopituitarism (group Hx), and the PCr level observed in rats HxG was similar to that calculated in T4-treated rats, due to a higher PCr/ATP ratio. The calculation of PP using the equilibrium equation of CK indicated a significantly lower value in rats maintained in hormonal deficiency than in the other three groups. 3.2. Results observed during the recovery period At the end of stimulation, the PCr depletion could be considered as equivalent in all groups of rats (Hx: 9.0 ± 3.6 mmol/kg, HxG: 9.2 ± 3.4 mmol/kg, HxT: 9.9 ± 2.4 mmol/kg, HxGT: 11.0 ± 2.6 mmol/kg) as well as the phosphorylation potential, which reached statistically equivalent values (Hx: 8.5 ± 5.2 kg M −1; HxG: 8.3 ± 5.0 kg M −1; HxT: 5.8 ± 1.8 kg M −1; HxGT: 6.3 ± 3.1 kg M −1). After the end of stimulation, the PCr recovered at a lower rate in T4-deprivated rats (groups Hx and HxG) than in the other two groups (p b 0.001 for the factor group without significant interaction factor). The calculation of the constant term T of the exponential model indicated that the rats treated with both hormones (group HxGT) exhibited a statistically higher PCr recovery rate than the groups Hx and HxG (Fig. 1). Since the pHi recovery was equivalent among the four groups and since the recovery of the phosphorylation potential could be considered as linear during the first 3 min (Fig. 2), analysis of the covariance was used to compare the slopes of PP, which was significantly higher in rats supplemented by the two hormones in comparison with the three other groups. 4. Discussion This study was undertaken in order to determine the respective impact of GH and T4 on energetic metabolism in skeletal muscle in vivo. Rats maintained in hypopituitarism exhibited lower resting PCr levels and lower PP than those treated with T4 or GH. Statistically equivalent concentrations at the end of muscle stimulation were
found in all experimental groups, permitting an evaluation of PCr recovery in similar experimental conditions. Thus, it is interesting to note that the high-phosphate metabolite levels varied differently, depending on whether the rats were treated with thyroxine or with growth hormone. Globally, we found that GH treatment given alone did not influence PCr recovery in hypophysectomized rats. Thyroxine treatment improved it, and this effect was more pronounced when GH was given simultaneously in rats, suggesting a potentiation of TH effects on mitochondrial function by GH administration. Indeed, the in vivo PCr recovery rate after exercise has a direct relationship with mitochondrial function [16,25]. Phosphocreatine regeneration occurs exclusively within the mitochondria and depends entirely on cell capacity for oxidative phosphorylation [26]. Although, it depends on PCr depletion and intracellular pH at the end of exercise, both of these parameters were found to be statistically equivalent in the four experimental groups in our study. Rats treated with thyroxine exhibited an increased post exercise PCr recovery rate, and these results are consistent with previous 31P MRS studies of hypothyroid rats or humans, in which decreased in vivo mitochondrial function was observed in hypothyroid states [18,19,27,28]. Oxidative phosphorylation accounts for more than 90% of the total energy yield and the stimulatory action of TH on the synthesis of enzymes of the respiratory chain has been well documented [29,30]. Respiratory chain subunits are encoded by nuclear genes, while others reside in the mitochondrial genome. There is probably a direct TH stimulatory effect of the regulatory agents on nuclear genes as well as on genes encoding mitochondrial transcription factors, which then secondarily affect mitochondrial transcription [2,10,30]. Indeed, TH receptors have been demonstrated in mitochondria [31,32] and the regulatory effect of thyroid hormone on mitochondrial transcription is partially exerted by a direct influence of the hormone on the mitochondrial transcription process [30]. Moreover, TH are also known to modulate the expression of heavy myosin isoform family genes. [33] hypothyroidism enhances the proportion of type I slow-twitch fibres, whilst TH induce a rise in type II fast-twitch fibres [34]. Moreover, an increase in mRNA content of cytochrome oxidase subunits or in mitochondrial substrate utilization by TH administration was essentially observed in slow-twitch oxidative muscle rather than in fast-twitch mixed muscle [35]. This could partly explain increased PCr recovery in thyroxine-treated rats. In contrast, the administration of GH did not significantly modify the recovery of PCr and of PP levels in T4-deprivated rats after the end of stimulation. These findings suggest that, in the absence of TH, the administration of GH does not significantly affect the oxidative phosphorylation. These results are consistent with a previous in vitro study, which demonstrated that hypophysectomized rats treated with both T4 and GH did not exhibit higher cytochrome oxidase activity than rats treated with T4 only [10]. We found an increased resting PCr value in the GH alone replaced rats, which contrasts with the low PCr synthesis rate after contractions. This may be due to an increase in creatine kinase activity induced by GH. This hypothesis is consistent with the findings of stimulated creatine kinase activity by GH in epiphyseal cartilage, kidney or liver [36], but requires further confirmation in skeletal muscle. Our study indicates that GH improves in vivo mitochondrial function in the presence of TH. A recent 31P.NMR study of healthy men
Table 1 Hormonal status in the 4 experimental groups at the time of NMR protocol. Abbreviations. Hx: hypopituitary rats; HxG: hypophysectomized rats treated with GH alone; HxT: hypophysectomized rats treated with thyroxine alone; HxGT: hypophysectomized rats treated with both GH and thyroxine.
−3
Initial weight (10 kg) Weight gain (10−3 kg) during hormone administration Free T3 (pmol/l) Free T4 (pmol/l) IGF (ng/ml)
Hx
HxG
HxT
HxGT
215 ± 7 −2 ± 4 0.42 ± 0.06 1.54 ± 0.25 6.28 ± 0.70
218 ± 6 −3 ± 4 0.43 ± 0.07 1.53 ± 0.30 9.00 ± 0.92
216 ± 9 26 ± 7 1.14 ±0.24 5.17 ± 0.93 5.90 ± 1.00
216 ± 9 28 ± 9 1.05 ± 0.24 5.01 ± 1.14 9.06 ± 1.14
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Table 2 Concentrations of ATP and total creatine, measured by reversed-phase HPLC, are presented with respect to the wet weight. Intracellular pH and the estimated concentrations of phosphocreatine (PCr) and inorganic phosphate (Pi) observed in the four experimental groups using 31P NMR spectroscopy are also given. The ratios of Pi/ATP, and PCr/ATP were calculated from the spectra using the ATPβ peak as reference for ATP level. The absolute concentrations were then estimated by multiplying these ratios by the value of ATP measured via RP-HPLC. The phosphorylation potential was estimated using the equilibrium of the CK reaction (see text for hypotheses). The last column indicates the result of the variance analysis. The symbols a,b,c, and d indicate a significant difference of the mean with respect to the value of the specific group Hx, HxG, HxT or HxGT (test of Tukey, p b 0.05). Abbreviations. Hx: hypopituitary rats; HxG: hypophysectomized rats treated with GH alone; HxT: hypophysectomized rats treated with thyroxine alone; HxGT: hypophysectomized rats treated with both GH and thyroxine.
ATP (mmol/kg) Total creatine (mmol/kg) pH Pi (mmol/kg) PCr (mmol/kg) PCr/Pi Phosphorylation potential (kg/M)
Hx n = 8
HxG n = 8
HxT n = 8
HxGT n = 8
Variance analysis
8.10 ± 0.46d 44.9 ± 1.6 7.09 ± 0.01b 4.00 ± 0.16 25.6 ± 0.8b,c,d 6.47 ± 0.30 48.2 ± 3.6b,c,d
7.41 ± 0.40c,d 43.9 ± 1.5 7.04 ± 0.01a,c,d 4.42 ± 0.23 32.1 ± 1.0a 7.36 ± 0.25 96.4 ± 8.3a
9.02 ± 0.33b 43.8 ± 0.6 7.09 ± 0.01b 4.43 ± 0.31 33.4 ± 0.5a 7.67 ± 0.36 98.2 ± 9.0a
9.75 ± 0.4a,b 45.9 ± 1.7 7.09 ± 0.01b 4.38 ± 0.22 34.0 ± 0.8a 7.94 ± 0.47 111.9 ± 17.0a
p b 0.02 n.s. p b 0.01 n.s. p b 0.001 n.s. p b 0.001
(who were by definition, euthyroid) who underwent GH stimulation testing with GH releasing hormone-arginine demonstrated that PCr recovery rate after submaximal exercise was associated with serum IGF-1, GH peak and GH area under the curve [17]. Acute effects of GH infusion on muscle mitochondrial function and gene transcripts have previously been established in healthy human subjects. [14] This included enhanced skeletal muscle mitochondrial ATP product,
Fig. 1. Comparison of phosphocreatine recovery after non-tetanic muscle stimulation in rats maintained in hypopituitarism (open triangles), in rats treated with GH alone (filled triangles), in rats treated with T4 (open squares) and in rats treated with both GH and T4 (filled squares). *: p b 0.05.
with higher muscle content of mRNA transcripts encoding oxidative proteins in mitochondria (COX 3 and COX4), a nuclear transcription factor that regulates mitochondrial biogenesis and the glucose transport protein GLUT4, while total muscle protein synthesis was unchanged within the 14 h GH infusion period [14]. This could be related to a direct GH effect or via IGF-I mediated effects on muscle and mitochondrial metabolism. The mechanisms through which GH action could potentiate TH-mediated mitochondrial substrate oxidation, as suggested by our results, remain to be elucidated. The fact that both GH and TH stimulate mitochondrial oxidative capacity and transcript levels of severalmitochondrial genes implies that the actions of these hormones might overlap [14]. However, GH and TH could also regulate mitochondrial function through different pathways [37], possibly acting in synergy. For example, GH action is known to activate proteins in the β-oxidation or tricarboxylic acid cycles with reduced carbohydrate utilization [14], while TH induce a shift in metabolic substrate utilization from carbohydrate to lipid oxidation [38]. Functional changes in the mitochondrial metabolism activity could also occur by modifying the ratio of unsaturated/saturated free fatty acids [39]. While TH induce substantial modifications in mitochondrial inner membrane protein and lipid compositions [40], growth hormone treatment increases mitochondrial oxidation of highly polyunsaturated fatty acids with a beneficial effect on mitochondrial membranes [41]. To conclude, this study demonstrates that hypopituitarism induces an oxidative metabolism defect in vivo. Only thyroid hormones act directly on the mitochondrial metabolism, while GH has no obvious effect if given alone, but nonetheless enhances the thyroxine effect, suggesting a synergistic action of T4 and GH.
Conflict of interest None.
References
Fig. 2. Phosphorylation potential recovery in the four groups of rats (same symbols as in Fig. 1). *: p b 0.05.
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Growth hormone potentiates thyroid hormone effects on post-exercise phosphocreatine recovery in skeletal muscle☆ P. Kaminsky a,⁎, P.M. Walker b, J. Deibener a, F. Barbe c, E. Jeannesson c, J.M. Escanye d, B. Dousset e, M. Klein f a
Pôle des Spécialités Médicales, Service de Médecine Interne, Centre Hospitalier Universitaire de Nancy, Hôpitaux de Brabois, 54500 Vandoeuvre, France Laboratoire de Biophysique, Faculté de Médecine-Pharmacie, Université de Bourgogne, 21000 Dijon, France Laboratoire de Biochimie des Protéines, Centre Hospitalier Universitaire de Nancy, Hôpitaux de Brabois, 54500 Vandoeuvre, France d Pôle Imagerie, Centre Hospitalier Universitaire de Nancy, Hôpitaux de Brabois, 54500 Vandoeuvre, France e Laboratoire de Biochimie, Centre Hospitalier Universitaire de Nancy, Hôpital Central, 54000 Nancy, France f Service d ' Endocrinologie, Centre Hospitalier Universitaire de Nancy, Hôpitaux de Brabois, 54500 Vandoeuvre, France b c
a r t i c l e
i n f o
Article history: Received 12 July 2011 Received in revised form 31 July 2012 Accepted 2 August 2012 Available online 28 August 2012 Keywords: Nuclear magnetic resonance Skeletal muscle Mitochondrion Somatotropin Thyroid hormones Rat
a b s t r a c t Objective: The aim of the study was to determine the respective impact of thyroxine and growth hormone on in vivo skeletal mitochondrial function assessed via post exercise phosphocreatine recovery. Design: The hind leg muscles of 32 hypophysectomized rats were investigated using 31P nuclear magnetic resonance spectroscopy at rest and during the recovery period following a non tetanic stimulation of the sciatic nerve. Each rat was supplemented with hydrocortisone and was randomly assigned to one of the 4 groups: the group Hx was maintained in hypopituitarism., the group HxT was treated with 1 μg/100 g/day of thyroxine (T4), the group HxG with 0.2 IU/kg/day of recombinant human GH (rGH) and the group HxGT by both thyroxine and rGH. Inorganic phosphate (Pi), phosphocreatine (PCr) and ATP were directly measured on the spectra, permitting the calculation of the phosphorylation potential (PP). Results: At rest, the rats treated with rGH or T4 exhibited higher PCr levels than rats Hx. The recovery rates of PCr and PP were higher in rats treated with T4 than in T4-deprivated rats, suggesting improved mitochondrial function. The rats treated by both T4 and rGH showed higher PCr and PP recovery than those maintained in hypopituitarism or treated with T4 or rGH alone. Conclusions: The study demonstrates that in contrast to T4, GH given alone in hypophysectomized rats does not improve in vivo mitochondrial oxidative metabolism. Growth hormone potentiates T4 effects on oxidative metabolism. © 2012 Elsevier Ltd. All rights reserved.
1. Introduction Growth hormone (GH) and thyroid hormones (TH) play an important role in cell growth and differentiation, and are important regulators of fuel metabolism in ATP-requiring processes. Mitochondria are the primary site of skeletal energetic metabolism and ATP production. At physiological levels, TH regulates numerous genes in skeletal muscles, especially those involved in energetic metabolism [1]. Not only does TH control the in vivo expression of a selected set of nuclear genes encoding mitochondrial inner membrane proteins [2], but they are also involved in mitochondrial DNA expression [3] encoding protein complex subunits in the respiratory chain.
☆ Funds: this work was supported by a grant of the Association Française contre les Myopathies. ⁎ Corresponding author at: Pôle des Spécialités Médicales, Service de Médecine Interne orientée vers les Maladies Orphelines et Systémiques, Centre Hospitalier Universitaire de Nancy, Hôpitaux de Brabois, rue du Morvan, 54500 Vandoeuvre-lès-Nancy, France. Tel.: +33 3 83 15 40 60; fax: +33 3 83 15 79 41. E-mail address: [email protected] (P. Kaminsky). 1096-6374/$ – see front matter © 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.ghir.2012.08.001
Moreover, the presence of both GH receptors [4] and IGF-I receptors [5] has been demonstrated in skeletal muscle. Growth hormone is known to act on protein anabolism, on the promotion of lipolysis, and on the resistance to insulin-induced glucose metabolism [6–8]. In animals, GH treatment does not modify mitochondrial respiratory rate in muscle or heart [9–11]. Furthermore, GH treatment was reported to reduce expression of several enzymes regulating lipid oxidation and energy production in hypopituitary men [12]. In contrast, an increased expression of a number of nuclear or mitochondrial genes involved in mitochondrial biogenesis was induced by GH [13–15]. In addition, in vivo post-exercise phosphocreatine (PCr) recovery rate, which is directly linked to mitochondrial function [16], has been reported recently to be correlated with IGF-1 and GH levels in healthy volunteers receiving GH stimulation with GH releasing hormone-arginine [17]. Such a discrepancy may be in part explained by functional metabolic changes induced in vivo by hormonal treatment. For instance, in muscle, the hypothyroid state induces a decreased activity of glycolysis in vitro, although in vivo studies have demonstrated an increased recruitment of this pathway, with enhanced lactate and H+ production due to decreased mitochondrial function [18,19]. In addition, hormonal status
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could be different in hypopituitary animals or patients among the different studies and little is known about the interaction between TH and GH at mitochondrial level. Using 31P magnetic resonance spectroscopy ( 31P.MRS), this study was aimed at evaluating the respective effects of GH and of TH on the in vivo mitochondrial function in hypophysectomized rats. 2. Material and methods 2.1. Animal care Male Sprague–Dawley adolescent rats (initial weight of approximatively 250 g) were purchased from Charles River France, housed four per cage in a temperature- and light-controlled room (22 °C; dark cycle 12 h–12 h), and provided with drinking water and standardized food ad libitum. They were hypophysectomized by the parapharyngeal method, and maintained in hypopituitarism for 3 weeks. All rats were treated with hydrocortisone (hydrocortisone, Roussel Laboratory, France, 62.5 μg/100 g twice daily) [20]. They were randomly assigned to four experimental groups of 8 rats: - the first group (labelled Hx) was maintained in hypopituitarism; - the second group (labelled HxT) was treated with substitutive doses of thyroxine (T4 : 1 μg/100 g/day) (L-Thyroxine¨, Laboratory Roche) [21]; - the third group (labelled HxG) was treated with 0.2 IU/kg/day of recombinant human GH (rGH : Genotonorm ¨, Laboratory Pfizer) [22]; - the fourth group (labelled HxGT) was treated by both thyroxine and rGH. Hydrocortisone and growth hormone were dissolved in saline and subcutaneously injected twice daily. Thyroxine was dissolved in saline and subcutaneously injected daily. All experiments were performed subsequent to an eleven-day hormonal treatment. Rats treated with rGH were all investigated 17 h ± 2 h after the last injection. Animal experiments were conformed to guidelines on animal care and use currently applied in our country. 2.2. Stimulation protocol The details of the stimulation and 31P.MRS protocol have previously been described elsewhere [23]. Briefly, animals were anaesthetized with ketamine (200 mg/kg, ip), with additional doses (50 mg/kg) given as needed to maintain deep anaesthesia during investigation. After exposing the right sciatic nerve, an insulated platinum bipolar electrode was placed around the exposed nerve. The animal was placed in the ventral position and the head of the gastrocnemius was positioned at the geometrical centre of the receiver coil (see below). The tendons of the muscles of the posterior compartment were then passively loaded with a weight of 100 g. The nerve stimulation (3 Hz) was achieved with rectangular supramaximal pulses (15 V, 1 ms duration), resulting in isometrical non-tetanic stimulation of the entire gastrocnemius–plantaris–soleus group. Muscle stimulation was maintained for 6 min, and immediately followed by the recording of a 10 min recovery period. This frequency level was chosen in order to obtain similar PCr depletion and intracellular pH in all the experimental groups by the end of stimulation.
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1994). Shimming of the static magnetic field was performed before each experiment using the 1H Free Induction Decay (FID). Forty eight FIDs were accumulated over 1 min with a repetition time (TR) of 1.25 s and over a bandwidth of 2000 Hz. The radiofrequency pulse length was 25 μs, and this corresponded to a maximum of signal for the 1.25 s TR. The resting spectra were obtained in 5 min in 2-koctet blocks of 240 FIDs. During the muscle stimulation and the post-stimulation recovery, the spectra were acquired continuously in 1-k octet blocks of 48 FIDs. Six spectra were hence recorded during the muscle stimulation and ten spectra during the recovery period with a time resolution of 60s/spectrum. The bone signal was filtered out by a 300 Hz differential convolution. Spectra were then apodized with 10 Hz of line broadening, Fourier transformed and phase-corrected, if required. After a second-order base-line correction, the peak area of PCr, phosphomonoester (PME), inorganic phosphate (Pi) and ATP were calculated by computed integration and corrected with the saturation factors obtained as described elsewhere [23]. 2.4. Biochemical assays After acquiring the NMR data, a muscle sample from the gastrocnemius was frozen in vivo, promptly dissected, immediately immersed in liquid nitrogen and stored at −80 °C. The levels of ATP and total creatine were assessed using reversed-phase high-performance liquid chromatography (RP-HPLC). The rat was then killed by exsanguination (aortic function), the blood sample being retained for control of hormonal assays (T3 and T4: Amersham International, London, UK; IGF-I: INCSTAR Co, Minnesota, USA). 2.5. Calculations The saddle coil collected the signal from all the hind-leg muscles below the knee. Thus, only the averaged absolute values in the hindleg were assessed. At rest, the ratios of PME/ATP, Pi/ATP, and PCr/ATP were calculated from the spectra using the ATPβ peak as reference for ATP level. The absolute resting concentrations were then estimated by multiplying these ratios by the value of ATP measured via RP-HPLC. During the muscle stimulation and recovery, the peak areas of phosphate metabolites were compared with that of a sample of diphosphonate (DP) strapped to the muscles within the saddle coil [23]. The time-course of PCr was directly calculated on the spectra through the ratio PCr/DP. We also estimated the phosphorylation potential (PP), defined as: PP ¼ ½ATP=ð½ADP ½PiÞ: It was calculated using the formula: PP ¼ ½PCr=½Pi ð½H þ KckÞ=ð½totalcreatine ½PCrÞ in which ATP and total creatine were considered as constant and measured in resting muscle using RP-HPLC, and the equilibrium constant Kck as equal to 1.66 10 9 M −1 [24]. The PCr recovery was modelized by a monoexponential fit, [PCr = A + B exp (− t / T)], the recovery rate being estimated using the constant exponential term T. The PP recovery was modelized using a linear regression during the first 3 min after the end of stimulation.
2.3. NMR protocol
2.6. Statistical analysis
The 31P.MRS experiments were performed with a 2.35 T, 40 cm diameter, horizontal superconducting magnet (Brüker BNT 100, Karlsruhe, Germany), using a saddle coil (18 mm in diameter×22 mm in length), which could be simultaneously tuned to the frequencies of both 1H (100.183 MHz) and 31P (40.5545 MHz) (Klein, Kaminsky et al.
All the results were obtained from 8 rats in each experimental group and are presented as group mean ± 1 S.D. in tables or text. In order to clarify the figures, the results are however shown as mean ± S.E., as specified in the legends. One-way analysis of variance was used to indicate an overall statistical significance among the
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means of the four experimental groups. Two-way analysis of variance (model of two factors with replication) was used to compare the time-evolution of the parameters during stimulation and recovery. The comparison of multiple linear regressions was tested using covariance analysis. Two-by-two comparisons were tested using the test of Tukey for multiple comparisons of means. A p-value of less than 0.05 was considered as significant. Statistical analyses were performed using the XLSTAT2009 statistical software package (Addinsoft). 3. Results 3.1. Results observed at rest Hormonal treatment resulted in a significant rise in the levels of thyroid hormone and IGF-I at the time of 31P.MRS investigation (Table 1). Only rats treated with T4 grew during the hormonal treatment. Table 2 shows the results obtained at rest in the four experimental groups. The concentration of ATP was significantly higher in all rats treated with T4 (groups HxT, and HxGT) than in T4-deprivated rats (Hx and HxG), while the levels of total creatine did not differ among the four groups. For 31P.MRS data, no differences were found for the Pi levels. However, PCr was lower in rats maintained in hypopituitarism (group Hx), and the PCr level observed in rats HxG was similar to that calculated in T4-treated rats, due to a higher PCr/ATP ratio. The calculation of PP using the equilibrium equation of CK indicated a significantly lower value in rats maintained in hormonal deficiency than in the other three groups. 3.2. Results observed during the recovery period At the end of stimulation, the PCr depletion could be considered as equivalent in all groups of rats (Hx: 9.0 ± 3.6 mmol/kg, HxG: 9.2 ± 3.4 mmol/kg, HxT: 9.9 ± 2.4 mmol/kg, HxGT: 11.0 ± 2.6 mmol/kg) as well as the phosphorylation potential, which reached statistically equivalent values (Hx: 8.5 ± 5.2 kg M −1; HxG: 8.3 ± 5.0 kg M −1; HxT: 5.8 ± 1.8 kg M −1; HxGT: 6.3 ± 3.1 kg M −1). After the end of stimulation, the PCr recovered at a lower rate in T4-deprivated rats (groups Hx and HxG) than in the other two groups (p b 0.001 for the factor group without significant interaction factor). The calculation of the constant term T of the exponential model indicated that the rats treated with both hormones (group HxGT) exhibited a statistically higher PCr recovery rate than the groups Hx and HxG (Fig. 1). Since the pHi recovery was equivalent among the four groups and since the recovery of the phosphorylation potential could be considered as linear during the first 3 min (Fig. 2), analysis of the covariance was used to compare the slopes of PP, which was significantly higher in rats supplemented by the two hormones in comparison with the three other groups. 4. Discussion This study was undertaken in order to determine the respective impact of GH and T4 on energetic metabolism in skeletal muscle in vivo. Rats maintained in hypopituitarism exhibited lower resting PCr levels and lower PP than those treated with T4 or GH. Statistically equivalent concentrations at the end of muscle stimulation were
found in all experimental groups, permitting an evaluation of PCr recovery in similar experimental conditions. Thus, it is interesting to note that the high-phosphate metabolite levels varied differently, depending on whether the rats were treated with thyroxine or with growth hormone. Globally, we found that GH treatment given alone did not influence PCr recovery in hypophysectomized rats. Thyroxine treatment improved it, and this effect was more pronounced when GH was given simultaneously in rats, suggesting a potentiation of TH effects on mitochondrial function by GH administration. Indeed, the in vivo PCr recovery rate after exercise has a direct relationship with mitochondrial function [16,25]. Phosphocreatine regeneration occurs exclusively within the mitochondria and depends entirely on cell capacity for oxidative phosphorylation [26]. Although, it depends on PCr depletion and intracellular pH at the end of exercise, both of these parameters were found to be statistically equivalent in the four experimental groups in our study. Rats treated with thyroxine exhibited an increased post exercise PCr recovery rate, and these results are consistent with previous 31P MRS studies of hypothyroid rats or humans, in which decreased in vivo mitochondrial function was observed in hypothyroid states [18,19,27,28]. Oxidative phosphorylation accounts for more than 90% of the total energy yield and the stimulatory action of TH on the synthesis of enzymes of the respiratory chain has been well documented [29,30]. Respiratory chain subunits are encoded by nuclear genes, while others reside in the mitochondrial genome. There is probably a direct TH stimulatory effect of the regulatory agents on nuclear genes as well as on genes encoding mitochondrial transcription factors, which then secondarily affect mitochondrial transcription [2,10,30]. Indeed, TH receptors have been demonstrated in mitochondria [31,32] and the regulatory effect of thyroid hormone on mitochondrial transcription is partially exerted by a direct influence of the hormone on the mitochondrial transcription process [30]. Moreover, TH are also known to modulate the expression of heavy myosin isoform family genes. [33] hypothyroidism enhances the proportion of type I slow-twitch fibres, whilst TH induce a rise in type II fast-twitch fibres [34]. Moreover, an increase in mRNA content of cytochrome oxidase subunits or in mitochondrial substrate utilization by TH administration was essentially observed in slow-twitch oxidative muscle rather than in fast-twitch mixed muscle [35]. This could partly explain increased PCr recovery in thyroxine-treated rats. In contrast, the administration of GH did not significantly modify the recovery of PCr and of PP levels in T4-deprivated rats after the end of stimulation. These findings suggest that, in the absence of TH, the administration of GH does not significantly affect the oxidative phosphorylation. These results are consistent with a previous in vitro study, which demonstrated that hypophysectomized rats treated with both T4 and GH did not exhibit higher cytochrome oxidase activity than rats treated with T4 only [10]. We found an increased resting PCr value in the GH alone replaced rats, which contrasts with the low PCr synthesis rate after contractions. This may be due to an increase in creatine kinase activity induced by GH. This hypothesis is consistent with the findings of stimulated creatine kinase activity by GH in epiphyseal cartilage, kidney or liver [36], but requires further confirmation in skeletal muscle. Our study indicates that GH improves in vivo mitochondrial function in the presence of TH. A recent 31P.NMR study of healthy men
Table 1 Hormonal status in the 4 experimental groups at the time of NMR protocol. Abbreviations. Hx: hypopituitary rats; HxG: hypophysectomized rats treated with GH alone; HxT: hypophysectomized rats treated with thyroxine alone; HxGT: hypophysectomized rats treated with both GH and thyroxine.
−3
Initial weight (10 kg) Weight gain (10−3 kg) during hormone administration Free T3 (pmol/l) Free T4 (pmol/l) IGF (ng/ml)
Hx
HxG
HxT
HxGT
215 ± 7 −2 ± 4 0.42 ± 0.06 1.54 ± 0.25 6.28 ± 0.70
218 ± 6 −3 ± 4 0.43 ± 0.07 1.53 ± 0.30 9.00 ± 0.92
216 ± 9 26 ± 7 1.14 ±0.24 5.17 ± 0.93 5.90 ± 1.00
216 ± 9 28 ± 9 1.05 ± 0.24 5.01 ± 1.14 9.06 ± 1.14
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Table 2 Concentrations of ATP and total creatine, measured by reversed-phase HPLC, are presented with respect to the wet weight. Intracellular pH and the estimated concentrations of phosphocreatine (PCr) and inorganic phosphate (Pi) observed in the four experimental groups using 31P NMR spectroscopy are also given. The ratios of Pi/ATP, and PCr/ATP were calculated from the spectra using the ATPβ peak as reference for ATP level. The absolute concentrations were then estimated by multiplying these ratios by the value of ATP measured via RP-HPLC. The phosphorylation potential was estimated using the equilibrium of the CK reaction (see text for hypotheses). The last column indicates the result of the variance analysis. The symbols a,b,c, and d indicate a significant difference of the mean with respect to the value of the specific group Hx, HxG, HxT or HxGT (test of Tukey, p b 0.05). Abbreviations. Hx: hypopituitary rats; HxG: hypophysectomized rats treated with GH alone; HxT: hypophysectomized rats treated with thyroxine alone; HxGT: hypophysectomized rats treated with both GH and thyroxine.
ATP (mmol/kg) Total creatine (mmol/kg) pH Pi (mmol/kg) PCr (mmol/kg) PCr/Pi Phosphorylation potential (kg/M)
Hx n = 8
HxG n = 8
HxT n = 8
HxGT n = 8
Variance analysis
8.10 ± 0.46d 44.9 ± 1.6 7.09 ± 0.01b 4.00 ± 0.16 25.6 ± 0.8b,c,d 6.47 ± 0.30 48.2 ± 3.6b,c,d
7.41 ± 0.40c,d 43.9 ± 1.5 7.04 ± 0.01a,c,d 4.42 ± 0.23 32.1 ± 1.0a 7.36 ± 0.25 96.4 ± 8.3a
9.02 ± 0.33b 43.8 ± 0.6 7.09 ± 0.01b 4.43 ± 0.31 33.4 ± 0.5a 7.67 ± 0.36 98.2 ± 9.0a
9.75 ± 0.4a,b 45.9 ± 1.7 7.09 ± 0.01b 4.38 ± 0.22 34.0 ± 0.8a 7.94 ± 0.47 111.9 ± 17.0a
p b 0.02 n.s. p b 0.01 n.s. p b 0.001 n.s. p b 0.001
(who were by definition, euthyroid) who underwent GH stimulation testing with GH releasing hormone-arginine demonstrated that PCr recovery rate after submaximal exercise was associated with serum IGF-1, GH peak and GH area under the curve [17]. Acute effects of GH infusion on muscle mitochondrial function and gene transcripts have previously been established in healthy human subjects. [14] This included enhanced skeletal muscle mitochondrial ATP product,
Fig. 1. Comparison of phosphocreatine recovery after non-tetanic muscle stimulation in rats maintained in hypopituitarism (open triangles), in rats treated with GH alone (filled triangles), in rats treated with T4 (open squares) and in rats treated with both GH and T4 (filled squares). *: p b 0.05.
with higher muscle content of mRNA transcripts encoding oxidative proteins in mitochondria (COX 3 and COX4), a nuclear transcription factor that regulates mitochondrial biogenesis and the glucose transport protein GLUT4, while total muscle protein synthesis was unchanged within the 14 h GH infusion period [14]. This could be related to a direct GH effect or via IGF-I mediated effects on muscle and mitochondrial metabolism. The mechanisms through which GH action could potentiate TH-mediated mitochondrial substrate oxidation, as suggested by our results, remain to be elucidated. The fact that both GH and TH stimulate mitochondrial oxidative capacity and transcript levels of severalmitochondrial genes implies that the actions of these hormones might overlap [14]. However, GH and TH could also regulate mitochondrial function through different pathways [37], possibly acting in synergy. For example, GH action is known to activate proteins in the β-oxidation or tricarboxylic acid cycles with reduced carbohydrate utilization [14], while TH induce a shift in metabolic substrate utilization from carbohydrate to lipid oxidation [38]. Functional changes in the mitochondrial metabolism activity could also occur by modifying the ratio of unsaturated/saturated free fatty acids [39]. While TH induce substantial modifications in mitochondrial inner membrane protein and lipid compositions [40], growth hormone treatment increases mitochondrial oxidation of highly polyunsaturated fatty acids with a beneficial effect on mitochondrial membranes [41]. To conclude, this study demonstrates that hypopituitarism induces an oxidative metabolism defect in vivo. Only thyroid hormones act directly on the mitochondrial metabolism, while GH has no obvious effect if given alone, but nonetheless enhances the thyroxine effect, suggesting a synergistic action of T4 and GH.
Conflict of interest None.
References
Fig. 2. Phosphorylation potential recovery in the four groups of rats (same symbols as in Fig. 1). *: p b 0.05.
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