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Am J Physiol Endocrinol Metab 294: E1127-E1134, 2008. First published April 15, 2008; doi:10.1152/ajpendo.00010.2008
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Effect of GH on human skeletal muscle lipid metabolism in GH deficiency

Roman Trepp,1 Martin Flück,3 Christoph Stettler,1 Chris Boesch,2 Michael Ith,2 Roland Kreis,2 Hans Hoppeler,3 Hans Howald,3 Jean-Paul Schmid,4 Peter Diem,1 and Emanuel R. Christ1

1Division of Endocrinology, Diabetology and Clinical Nutrition, Inselspital, Bern University Hospital and University of Bern; 2Department of Clinical Research (Magnetic Resonance Spectroscopy and Methodology), University of Bern; 3Department of Systematic Anatomy; and 4Cardiovascular Prevention and Rehabilitation, Department of Cardiology, Inselspital, University of Bern, Bern, Switzerland

Submitted 4 January 2008 ; accepted in final form 13 April 2008


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Adult-onset growth hormone (GH) deficiency (GHD) is associated with insulin resistance and decreased exercise capacity. Intramyocellular lipids (IMCL) depend on training status, diet, and insulin sensitivity. Using magnetic resonance spectroscopy, we studied IMCL content following physical activity (IMCL-depleted) and high-fat diet (IMCL-repleted) in 15 patients with GHD before and after 4 mo of GH replacement therapy (GHRT) and in 11 healthy control subjects. Measurements of insulin resistance and exercise capacity were performed and skeletal muscle biopsies were carried out to assess expression of mRNA of key enzymes involved in skeletal muscle lipid metabolism by real-time PCR and ultrastructure by electron microscopy. Compared with control subjects, patients with GHD showed significantly higher difference between IMCL-depleted and IMCL-repleted. GHRT resulted in an increase in skeletal muscle mRNA expression of IGF-I, hormone-sensitive lipase, and a tendency for an increase in fatty acid binding protein-3. Electron microscopy examination did not reveal significant differences after GHRT. In conclusion, variation of IMCL may be increased in patients with GHD compared with healthy control subjects. Qualitative changes within the skeletal muscle (i.e., an increase in free fatty acids availability from systemic and/or local sources) may contribute to the increase in insulin resistance and possibly to the improvement of exercise capacity after GHRT. The upregulation of IGF-I mRNA suggests a paracrine/autocrine role of IGF-I on skeletal muscle.

intramyocellular lipids; insulin resistance; magnetic resonance spectroscopy; exercise capacity

LOCALLY STORED LIPIDS WITHIN the skeletal muscle, so-called intramyocellular lipids (IMCL), have been shown to depend on training status, diet, and insulin sensitivity (39). In endurance-trained athletes, IMCL concentrations are increased compared with lean sedentary individuals and correlate positively with indexes of insulin sensitivity (9, 24, 35). Intriguingly, IMCL content is also enlarged in sedentary patients with insulin resistance (obesity and/or diabetes mellitus Type 2), and in these subjects IMCL concentrations correlate positively with indexes of insulin resistance (17, 22, 24, 35). This apparent paradox might be explained by insulin sensitivity depending on the capacity to utilize IMCL rather than on the absolute IMCL content (20, 36). Accordingly, depletion of IMCL stores during exercise is much less pronounced in patients with Type 2 diabetes compared with trained athletes (20, 36). The underlying mechanisms may be different expression of enzymes involved in skeletal muscle lipid metabolism resulting in an increase in lipid uptake and utilization (11).

In healthy subjects, an acute bolus of growth hormone (GH) results in an increase in nonesterified free fatty acid (NEFA) concentrations with a concomitant increase in systemic lipid oxidation (21). In addition, microdialysis studies demonstrated an increase in lipolysis in adipose tissue (10). In parallel, GH reduces uptake and oxidation of glucose in skeletal muscle signifying an increase in insulin resistance (18). In patients with GH deficiency (GHD), long-term GH replacement therapy (GHRT) leads to a reduction in fat mass, predominately in the visceral compartment, further corroborating the strong lipolytic effect of GH (7).

The consequences of the GH-induced lipolysis at the skeletal muscle level are not fully elucidated. Theoretically, systemically available lipids [VLDL-triglycerides (TG) and NEFA] and locally stored lipids (IMCL) are potential targets for GH action. In healthy subjects, high-dose GH therapy resulted in an increase in IMCL in parallel with an increase in insulin resistance, fat oxidation, and NEFA turnover but unchanged VLDL-TG turnover (21). These findings suggest that GH influences fat oxidation in skeletal muscle and that circulating NEFA rather than VLDL-TG are the major source for lipid oxidation. In addition, IMCL or their degradation products may be involved in GH-induced insulin resistance.

In patients with GHD, the direct action of GH on lipid uptake, trafficking, utilization, and regulation at the skeletal muscle level is currently unknown. In the present study, the primary end point was to investigate IMCL content in relation to physical activity and diet in hypopituitary patients with GHD before and after GHRT using magnetic resonance spectroscopy (MRS). In addition, these data were compared with identical measurements in sedentary healthy control subjects, matched for age, sex, and body mass index (BMI). To investigate underlying mechanisms (secondary end points), skeletal muscle ultrastructure, in particular size and number of mitochondria, was analyzed by electron microscopy and expression of mRNA of key enzymes involved in skeletal muscle lipid uptake, intracellular trafficking, utilization, and regulation was assessed by real-time PCR technique. We hypothesized that skeletal muscle IMCL are low in GHD compared with control subjects and that GHRT would increase IMCL. Furthermore, it is speculated that GHRT increases proteins involved in free fatty acid uptake [e.g., fatty-acid binding protein 3 (FABP3)] and IMCL degradation [e.g., hormone-sensitive lipase (LIPE)].


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Study subjects. Fifteen patients with adult-onset GHD for at least 12 mo (5 women and 10 men, age 42 ± 12 yr, BMI 27.0 ± 2.6 kg/m2, mean ± SD) and 11 healthy control subjects matched for age and BMI (3 women and 8 men, age 39 ± 9 yr, BMI 25.1 ± 3.0 kg/m2, mean ± SD, P value for age and BMI not significant vs. patients) were recruited between October 2002 and July 2005 at the Division of Endocrinology and Diabetes at the University Hospital of Bern, Switzerland. GHD was defined as a peak GH of less than 3 mU/l during an insulin provocation test with nadir plasma glucose of less than 2.2 mmol/l and hypoglycemic symptoms (34). Patients were included provided they had been under stable conventional hormone replacement therapy (glucocorticoid, thyroidal, and gonadal) as needed for at least 6 mo. Exclusion criteria were (former or present) ACTH- or GH-secreting pituitary adenoma, abnormal liver or renal function, active neoplasia, severe cardiovascular disease (unstable coronary artery disease, heart failure New York Heart Association III–IV), diabetes mellitus, hemophilia, therapy with drugs known to affect lipid or glucose metabolism, inability to exercise, and contraindications to exposure to a 1.5-T magnetic field. Nineteen patients with adult-onset GHD were recruited. Three patients had to be excluded because of nonadherence to medications and/or study protocol and one patient was excluded because of insufficiently substituted hypothyroidism at time of investigation. Eleven control subjects were investigated once, and all of them completed the study.

The study was performed according to the declaration of Helsinki, the guidelines of good clinical practice, the Swiss health laws, and the ordinance on clinical research. Approval for the study was obtained from the Ethics Committee Bern. Each study subject gave written, informed consent.

Study protocol. The study protocol is outlined in Fig. 1. The identical protocol was performed in patients (at baseline and after 4 mo of GHRT) and control subjects (studied only once). At the first visit, spiroergometry was carried out by using an electronically braked bicycle ergometer with determination of peak aerobic capacity. Subjects had fasted for at least 8 h and restrained from strenuous activity for at least 24 h. The usual replacement therapy was administered prior to exercise. Male patients with substitution of the gonadal axis were studied in the middle of the dose interval of testosterone injection. During the following 3 days, the patients exercised (walking) at a heart rate corresponding to 50% of maximum oxygen consumption (VO2 max) for 1 h per day. This intensity has been shown to significantly reduce IMCL levels (4, 30). Exercise compliance was monitored by use of a pulse watch (Polar) during the time of exercise and a pedometer (Digi-Walker) throughout the 3 days to assess physical activity. The patients and control subjects were asked to aim for >10,000 paths/day during the depletion period. During these 3 days, the patients followed a low-fat diet. Food diary and pedometer count were kept to repeat the same physical activity and diet 4 mo after GHRT. Food diary and physical activity of the control subjects were matched with the information of the corresponding patient.


Figure 1
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  		Fig. 1. Study protocol.

 
At visit 2, measurement of depleted IMCL stores was performed. The patients attended the hospital after an overnight fast (at least 8 h fasting). Basal metabolic rate was assessed by indirect calorimetry, and blood samples were taken to measure plasma concentrations of hormones (IGF-I, insulin) and metabolites (lipid profile, NEFA, glucose). Usual medication was administered. Subsequently, IMCL concentrations in the tibialis anterior muscle were measured using 1H-MRS. Immediately following the MRS, skeletal muscle biopsy was performed.

To replenish IMCL stores, the patients were asked to refrain from physical activity (aim: 5,000 paths/day) during 3 days. In addition, instructions for a high-fat diet during this period were given at the end of visit 2. This consisted of the usual food intake with a supplementary fat intake of 0.75 g fat/kg body wt per day, administered as three additional fat-enriched snacks provided by the research team. Three types of snacks were offered: Ragusa 50-g chocolate nut bar (21 g fat; 42% saturated fatty acids), butter croissant (8 g fat; 58% saturated fatty acids), and 100 g of peanuts (50 g fat; 20% saturated fatty acids). The typical three snacks for a 70-kg patient were one butter croissant in the morning (8 g fat), a 50-g chocolate-nut Ragusa bar (21 g fat) in the afternoon, and 50 g of peanuts in the evening (25 g fat). After 3 days of high-fat diet and low physical activity, MRS for IMCL of the tibialis muscle was repeated (visit 3). The time period was chosen because recent data have shown that in trained and untrained subjects repletion of skeletal IMCL occurs within 30 h (3). Food diary and pedometer count were kept to repeat the same physical activity and diet 4 mo after GHRT. Food diary and physical activity of the control subjects were matched with the information of the corresponding patient.

Patients with GHD were instructed in self-administration of GH using a pen device (Genotropin-Pen, Pfizer, Switzerland). Usual clinical care was provided (monthly visit with bioimpedance and IGF-I measurements to adjust GH doses). GH dose was gradually increased to obtain IGF-I concentrations in the upper half of the age-adjusted reference range as suggested by the Growth Hormone Research Society (34). Target IGF-I levels were obtained after 2–3 mo in all patients. After a total of 4 mo of GHRT, weight-maintaining diet, and usual physical activity, studies identical to visits 1-3 were performed. Diet protocols, fat snacks, and pedometer records were used to match diet and physical activity of the first study.

Spiroergometry. VO2 max and oxygen consumption at the ventilatory threshold (VO2VT) were determined during an incremental workload test to exhaustion with an electronically braked bicycle ergometer (Ergoline, Pilger, St. Gallen, Switzerland). Continuous analysis of expired oxygen, carbon dioxide content, and minute ventilation using a breath-by-breath analysis (Oxycon alpha, Jaeger, Würzburg, Germany) was performed. The subjects began at a workload of 20 W, which was gradually increased by 10–20 W every minute until exhaustion. The increase in workload was chosen to obtain an exercise time of 10–12 min in these sedentary subjects.

Indirect calorimetry and anthropometric measurements. Basal metabolic rate was assessed by indirect calorimetry using Deltatrac II (AVL Medical Systems, Schaffhausen, Switzerland) after an overnight fast in a quiet room during 20 min at a temperature of 22°C.

Body weight was measured on an electronic balance with subjects wearing light clothes and no shoes. Height was assessed by a stadiometer. BMI was calculated as the weight divided by the square of the height.

Biochemical analysis and calculations. Serum IGF-I was measured by an immunoradiometric assay (Nichols Institute, San Juan Capistrano, CA). Plasma glucose was measured by the hexokinase method (Hitachi 917, Roche, Basel, Switzerland). Insulin was determined by radioimmunoassay (Linco Research, Labodia, Yens, Switzerland) and HbA1c by capillary electrophoresis. TG were determined by use of commercially available kits (enzymatic method, Boehringer Mannheim, Mannheim, Germany) on a Hitachi 917 (Roche, Basel). Plasma NEFA levels were determined by enzymatic colorimetric procedure (Wako Chemicals, Freiburg, Germany). The quantitative insulin sensitivity check (QUICKI) indexes were calculated as 1/[log fasting insulin concentration (mU/l) + log fasting glucose concentration (mg/dl)] (19).

Magnetic resonance spectroscopy. IMCL stores were measured in the right tibialis anterior muscle by MRS, which was performed on a 1.5-T whole body system (Signa, General Electrics, Milwaukee, WI). The tibialis anterior muscle was chosen because the orientation of the fibers is nearly parallel to the magnetic field, allowing for an optimal separation of the IMCL resonance (4, 9, 31). Moreover, GH-deficient patients are usually sedentary patients and the best way to exercise in sedentary patients is walking. Walking, in turn, uses tibialis anterior muscle much more than quadriceps muscle, thereby depleting IMCL. In addition, IMCL depletion in larger skeletal muscles can easily be limited by systemic cardiovascular factors (3). Images and spectra were acquired with a 1H flexible coil (Medical Advance; Helmholtz design). IMCL concentrations levels were measured in a single 11 x 12 x 18 mm3 voxel in the middle of the right tibialis anterior muscle by using an optimized press sequence (repetition time = 3 s, echo time = 20 ms, 128 acquisitions, water presaturation, outer volume suppression) and quantified by using the fully relaxed, unsuppressed water signal as internal concentration standard. Absolute IMCL levels in millimoles per kilogram muscle wet weight were calculated as reported earlier (3).

Isolation and analysis of skeletal muscle biopsies. Biopsies were harvested (in the depleted state) from the middle portion of the right tibialis anterior muscle under local anesthesia by the Bergstroem technique (23). Electron microscopic morphometric determination of fiber-type distribution, fiber size, capillarity, and ultrastructural composition of muscle fibers was performed as described earlier (14, 15). Quantification of transcript expression was carried out as described previously (12, 13, 37). Total RNA was isolated from the frozen cross sections of biopsies by using the RNeasy minikit (Qiagen, Basel, Switzerland) and quantified with the RiboGreen RNA quantification kit (Molecular Probes, Juro, Lucerne, Switzerland). Six hundred nanograms of RNA were reverse transcribed into cDNA by using random hexamer primers with the Omniscript reverse transcriptase kit (Qiagen). Volumina of the cDNA product corresponding to 6 ng initial RNA were subjected to real-time PCR amplification on an ABI Prism 5700 sequence detection system with probe detection via SYBRgreen (PE Biosystems, Rotkreuz, Switzerland). Primer pairs for target mRNA (Table 1) were designed with the Primer Express software (PE Biosystems). The relative RNA amount of IGF-I, peroxisome proliferator-activated receptor-{alpha} and -{gamma}, lipoprotein lipase, LIPE, FABP3, adipocyte lipid-binding protein, cytochrome c oxidase 1 and 4, glucose transporter 4, and phosphofructokinase were estimated with the cycle threshold method for the amplification efficiency of the individual primer pairs for a given amount of reverse-transcribed RNA. The RNA estimates were then normalized to the mean of values for the respective transcript prior to GHRT.


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  		Table 1. PCR primers

 
Statistical analysis. On the basis of a publication comparing insulin-resistant individuals with healthy control subjects using MRS to measure IMCL (17), a sample size of 13 individuals per group is required to obtain conventional statistical significance (P = 0.05) with a power of 80%. We, therefore, decided to include 15 patients. Differences in continuous outcomes were investigated by Wilcoxon's signed-ranks test and the Mann-Whitney U-test for paired and unpaired data, respectively. Nonparametric Spearman rank correlations between IMCL concentrations and different markers of fasting metabolism and between mRNA transcripts were analyzed. A two-sided P value of <0.05 was considered significant. Data are presented as means ± SD. Analyses were performed by use of GraphPad InStat version 3.00, GraphPad Software, San Diego, CA.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Clinical characteristics anthropometry, basal metabolic rate, and exercise capacity. Clinical characteristics of GHD patients are shown in Table 2. Anthropometry, basal metabolic rate, and exercise capacity (maximal ventilatory oxygen uptake = VO2 max; ventilatory oxygen uptake at the anaerobic threshold = VO2VT, and heart rate at 50% of VO2 max) of GHD patients before and after GHRT and of control subjects are summarized in Table 3. Patients and control subjects did not differ in terms of age, sex, weight, BMI, and basal metabolic rate. Compared with control subjects GHD patients had a lower VO2 max, VO2VT, and heart rate at 50% VO2 max. VO2VT significantly increased following GHRT. After GHRT, VO2 max, VO2VT, and heart rate at 50% VO2 max remained lower compared with control subjects.


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  		Table 2. Clinical characteristics of patients

 

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  		Table 3. Anthropometry, calorimetry, and exercise capacity

 
Pedometer counts per day during depletion and repletion did not significantly differ in patients with GHD before and after GHRT and control subjects (GHD depletion: 13,689 ± 4,549; GHD repletion: 5,717 ± 1,929; GHRT depletion 14,002 ± 5,345; GHRT repletion 6,564 ± 1,988 and control depletion: 13,976 ± 1,473, control repletion: 5,973 ± 1,018).

Compared with control subjects, patients with GHD had lower IGF-I levels (87 ± 50 vs. 150 ± 47 ng/ml, means ± SD; P = 0.003) and a higher waist circumference (P = 0.036).

GHRT resulted in an increase in IGF-I concentrations (+98 ± 66 ng/ml, P < 0.001) and a decrease in waist circumference (–1.6 ± 2.3 cm, P = 0.03).

Compared with control subjects, patients after GHRT had similar IGF-I levels and waist circumference.

Biochemical results. Table 4 summarizes the biochemical results. Compared with control subjects, patients with GHD had similar fasting glucose, insulin, NEFA levels, and QUICKI indexes. Fasting TG were increased in patients with GHD compared with control subjects (P = 0.003).


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  		Table 4. Fasting biochemical results

 
GHRT did not result in significant changes in fasting glucose, insulin, NEFA, and TG concentrations and QUICKI indexes.

Compared with control subjects, patients after GHRT had similar fasting glucose, insulin, QUICKI, and NEFA concentrations. TG levels were increased in patients compared with control subjects (P = 0.006).

Intramyocellular lipids. Figure 2 shows depleted and repleted IMCL concentrations. Depleted IMCL were lower than repleted IMCL in control subjects (1.880 ± 1.635 vs. 2.935 ± 0.986 mmol/kg wet wt, P = 0.032), in patients with GHD before GHRT (1.579 ± 0.765 vs. 4.039 ± 1.457 mmol/kg wet wt, P < 0.001) and after GHRT (2.121 ± 1.259 vs. 4.043 ± 1.744 mmol/kg wet wt, P < 0.001).


Figure 2
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  		Fig. 2. Intramyocellular lipids (IMCL; means ± SD) after depletion by physical exercise and after repletion by high-fat diet, respectively, measured by magnetic resonance spectroscopy in patients with growth hormone deficiency before (GHD) and after growth hormone replacement therapy (GHRT) and in healthy control subjects (Control).

 
Compared with control subjects, patients with GHD had similar depleted IMCL but repleted IMCL showed a tendency to be higher (P = 0.072). GHRT did not significantly affect depleted and repleted IMCL. Compared with control subjects, patients after GHRT had similar depleted and repleted IMCL.

Figure 3 shows differences between depleted and repleted IMCL (delta-IMCL). Compared with control subjects, patients with GHD showed significantly higher delta-IMCL (1.922 ± 1.101, mmol/kg wet wt, P < 0.001). GHRT did not significantly affect delta-IMCL. Compared with control subjects, patients after GHRT had still significantly higher delta-IMCL (P = 0.026).


Figure 3
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  		Fig. 3. Differences between depleted and repleted IMCL (means ± SD) measured by magnetic resonance spectroscopy in patients with growth hormone deficiency before and after growth hormone replacement therapy and in healthy control subjects.

 
No significant correlations between IMCL levels and the markers of fasting metabolism could be detected (results not shown).

Messenger RNA transcripts in the tibialis anterior muscle. GHRT-induced changes in mRNA transcripts are shown in Fig. 4. GHRT induced an increase in mRNA of IGF-I (+130 ± 233%, P = 0.026), LIPE (+336 ± 489%, P = 0.011), and FABP3 (+30 ± 64%, P = 0.080), although the latter did not reach conventional level of significance.


Figure 4
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  		Fig. 4. Messenger RNA transcripts (means ± SD normalized to the respective mean of values prior to GHRT, i.e., GHD = 1) of IGF-I, peroxisome proliferator-activated receptor (PPAR) {alpha} and {gamma}, lipoprotein lipase (LPL: transport of free fatty acids from endothelium to the myocellular compartment), hormone-sensitive lipase (LIPE: degradation of IMCL), muscle fatty acid-binding protein (FABP3: uptake of free fatty acids), adipocyte lipid-binding protein (FABP4), cytochrome c oxidase (COX) 1 and 4, glucose transporter-4 (GLUT4: facilitates glucose uptake) and phosphofructokinase (PFK: key enzyme in the glycolytic pathway) in the tibialis anterior muscle of patients with growth hormone deficiency before and after growth hormone replacement therapy.

 
In patients with GHD, positive correlations were found for mRNA levels of IGF-1, LIPE, and FABP3 with each other (P < 0.05 for all correlations). After GHRT, positive correlation between mRNA of IGF-I and LIPE disappeared whereas the correlations between IGF-I and FABP3 and between LIPE and FABP3 remained stable.

Differences in mRNA expression of FABP3 before and after GHRT (delta-FABP3 mRNA) showed a strong positive correlation with delta-LIPE mRNA (r = 0.7, P = 0.006) and a trend to positively correlate with delta-IGF-I mRNA (r = 0.51, P = 0.078).

Ultrastructural adjustments in the tibialis anterior muscle. Ultrastructural examination did not reveal significant differences between patients with GHD before and after GHRT with regard to fiber size, capillary length, capillary-to-fiber ratio, volume density of total mitochondria, and volume density of subsarcolemmal mitochondria.


   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The main results of this study are that 1) variation of IMCL (delta between depletion and repletion) is increased in GHD patients compared with control subjects; 2) GHRT resulted in an increase in LIPE and a tendency for an increase in FABP3 mRNA expression; and 3) GHRT induced an increase in skeletal muscle IGF-I mRNA expression.

Increased variation of IMCL in GHD patients. Patients and control subjects showed differences between depleted and repleted IMCL content following physical activity and diet, consistent with earlier findings in different populations of patients and control subjects (3, 4, 9, 17, 21, 22, 24, 35, 36, 39). Interestingly, delta-IMCL was increased in GHD patients compared with control subjects, suggesting that GHD patients have an increased capacity to utilize and refill IMCL, which may be associated with an increased insulin sensitivity as shown in athletes (20, 36). However, insulin sensitivity was similar in GHD patients and control subjects, and no significant correlation between IMCL levels, delta-IMCL, and insulin sensitivity could be detected. These results indicate that in GHD patients insulin sensitivity is not the only parameter that regulates IMCL and its utilization. In healthy subjects Krag et al. (21) have recently shown that GH therapy results in an increase in IMCL in parallel with a decrease in insulin sensitivity. This is in contrast to our findings. Most likely these results relate to the fact that in this study healthy subjects were treated during 8 days with a high dose of GH (2 mg) inducing a decrease in insulin sensitivity. Furthermore, assessment of the metabolic flexibility (depletion-repletion) was not included in the protocol (21), and, finally, insulin sensitivity was assessed by a hyperinsulinemic euglycemic clamp, which is the gold standard to measure insulin sensitivity at the skeletal muscle level. Interestingly, Krag et al. could not detect significant correlations between IMCL and measurements of insulin sensitivity before and after GH therapy (21), in keeping with the present results.

The IMCL contents in the depleted and repleted state in patients with GHD before and after GHRT were approximately half the concentrations compared with endurance-trained athletes (39). In endurance-trained athletes, locally stored IMCL are an important source for energy during exercise and are related to exercise capacity (39). Moreover, exercise is a potent physiological stimulus for GH secretion (38) and high doses (2 mg per day) of GH during 8 days have recently shown to increase IMCL in healthy subjects (21). Therefore, it can be speculated that higher (supraphysiological) GH doses and/or concomitant regular physical training would have been necessary to increase IMCL levels in patients with GHD.

Increased expression of LIPE and FABP3 mRNA after GHRT. The increase in mRNA expression of LIPE and the tendency for an increase in FABP3 following GHRT in the present study suggests an improved availability of free fatty acids as a source of fuel within the skeletal muscle. Accordingly, recent evidence indicates that the main difference between trained and untrained healthy subjects within the skeletal muscle is an upregulation of mRNA of LIPE and FABP3 (32), implying that these proteins may be critical for exercise performance.

The GH-induced impairment of insulin sensitivity is traditionally speculated to be secondary to increased lipid availability in target tissues such as skeletal muscle (Randle cycle; 1, 16, 26, 27). Recent publications further substantiated this concept, reporting an increase in insulin sensitivity accompanied by a decrease in lipolysis in GH-deficient patients following combined therapy with GH and acipimox (28, 33). Acipimox is known to suppress lipolysis by inhibiting the activity of LIPE (8). Nonetheless, in the present study insulin sensitivity only tended to decrease following GHRT. Thus the relation between LIPE upregulation and insulin sensitivity after GHRT at the skeletal muscle level remains speculative.

FABP3 is an abundant cytosolic protein involved in NEFA uptake and utilization (2). To date, only results from rat studies are available, suggesting that GH increases the expression of FABP3 of the liver (6). In the present study, GHRT tended to increase mRNA of FABP3 in skeletal muscle, indicating that GH may increase NEFA uptake. This hypothesis is further corroborated by a recent report showing that GH therapy increases NEFA flux (and not VLDL-TG uptake) in healthy subjects (21).

Increased expression of skeletal muscle IGF-I mRNA after GHRT. Skeletal muscle IGF-I mRNA expression significantly increased following GHRT, indicating that the effects of GHRT on skeletal muscle may be mediated not only by systemically available IGF-I but also by locally synthesized IGF-I. Consistent with these findings, low-dose GH therapy in healthy elderly men resulted in an increase in skeletal muscle IGF-I expression (5). The tendency for a significant correlation between IGF-I expression and FABP-3 may indicate that changes in lipid metabolism may be mediated by a paracrine/autocrine function of IGF-I. Furthermore, it signifies that the GH-IGF-I axis may not only impact on myogenic differentiation and repair by modulating protein metabolism (5) but may also be involved in regulating skeletal muscle lipid muscle metabolism.

The strength of this study is the comprehensive assessment of the effect of GH on skeletal muscle lipid metabolism (in the depleted and repleted state) using metabolic parameters, MRS as well as morphological and functional analysis of skeletal muscle biopsies. However, several limitations of this study should be acknowledged: 1) The number of patients was relatively small because of the extensive investigations. Although an ideal patient group for studying GH actions would consist of subjects with isolated GHD, these patients are extremely rare in adulthood and do not reflect the majority of adult patients receiving GHRT. 2) Other skeletal muscles than the tibialis anterior muscle may show different behavior with regard to fuel metabolism. The fact that depletion and repletion were possible in all patients and control subjects indicates that the tibialis anterior muscle responds to diet and physical activity and is an adequate muscle to investigate in these patients. 3) The study protocol was based on experience in sedentary subjects aiming at completely deplete and replete IMCL in a standardized way (9). Hence, it did not allow to detect the velocity by which IMCL were utilized or repleted by exercise and diet and to separately assess the impact of diet and exercise on IMCL. 4) We cannot exclude that the additional measurements of IMCL before the depletion period would have eliminated a possible confounding factor.

In conclusion, this study suggests that variation of IMCL may be increased in patients with GHD compared with healthy control subjects. IMCL levels in patients with GHD are not increased by GHRT at recommended doses aiming at IGF-I levels in the upper half of the age-adjusted reference range. Qualitative changes within the skeletal muscle [i.e., an increase in free fatty acids availability from systemic (FABP3) and/or local (LIPE) sources] may contribute to the increase in insulin resistance and possibly to the improvement of exercise capacity after GHRT. The role of the upregulation of IGF-I mRNA suggests a paracrine/autocrine role of IGF-I. How long-term GHRT or a combination of regular physical exercise with GHRT will modify skeletal muscle lipid metabolism in patients with GHD remains to be established.


   GRANTS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
The study was funded by grants from the Swiss National Foundation to Emanuel R. Christ (no. 32000B0-100146) and to Chris Boesch (no. 3100A0-105815). Christoph Stettler is a PROSPER fellow supported by the Swiss National Science Foundation (Grant no. 3233B0 115212). Pfizer AG Switzerland kindly provided GH for this study.


   ACKNOWLEDGMENTS
 
We thank all the enthusiastic patients and control subjects who agreed to participate in this study.


   FOOTNOTES
 

Address for reprint requests and other correspondence: E. R. Christ, Division of Endocrinology and Diabetes, Inselspital, Univ. of Bern, 3010 Bern, Switzerland (e-mail: emanuel.christ{at}insel.ch)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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