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Original Studies |
Medical Department M (Endocrinology and Diabetes) (S.F., J.S.C., J.O.L.J.), Department of Endocrinology and Metabolism (K.K., S.B.P., B.R.), and Institute of Experimental Clinical Research (L.E.), Aarhus University Hospital, DK-8000 Aarhus; and Department of Internal Medicine and Endocrinology, Hvidovre University Hospital (A.M.R., J.H.), Copenhagen, Denmark
Address all correspondence and requests for reprints to: Sanne Fisker, M.D., Ph.D., Medical Department M, Aarhus Kommunehospital, Nørrebrogade 44, DK-8000 Aarhus C, Denmark. E-mail: sanne.fisker{at}dadlnet.dk
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Abstract |
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We examined the gene expression of GHR and GHRtr in human adipose tissue and skeletal muscle and the influence of GH treatment on this expression. Furthermore, we studied the relationship of circulating GHBP and body composition to GHR and GHRtr gene expression.
Eleven adult GH-deficient patients were studied before and after 4 months of GH substitution therapy. Abdominal fat obtained by liposuction and femoral muscle biopsies were taken at baseline and after 4 months. Gene expression of GHR and GHRtr in adipose tissue and skeletal muscle was determined and expressed relative to the expression of ß-actin.
Gene expression of GHR in abdominal sc adipose tissue was not altered, whereas the expression of GHRtr increased significantly. In skeletal muscle inverse changes were seen in the expression of messenger ribonucleic acid (mRNA) levels for the two GH receptor forms: expression of GHR increased significantly, whereas mRNA levels for GHRtr decreased. As expected, body composition changed with reduction of body fat mass after 4 months of GH treatment. Levels of circulating GHBP decreased significantly.
We conclude that GH treatment in GH-deficient adults changes the expression of mRNA for GHR and GHRtr in adipose tissue and skeletal muscle. Whether these changes are responsible for the observed changes in body composition in response to GH treatment and the observed changes in levels of circulating GHBP, however, needs further elucidation.
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Introduction |
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TopAbstractIntroductionSubjects and MaterialsResultsDiscussionReferences |
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Circulating GHBP is produced by cleavage of the extracellular part of the GH receptor (2), whereas in rodents GHBP it is produced from an alternatively spliced mRNA encoding for the extracellular domain of the receptor (3). It has been shown that cleavage of the extracellular part of the GH receptor in humans takes place in several tissues and from at least two different forms of the receptor (1). In previous studies we found elevated levels of GHBP, which decreased after GH treatment in GH-deficient patients (4, 5). We previously suggested that circulating GHBP may partly originate from adipose tissue, as circulating GHBP levels are strongly correlated to specific measures of adipose tissue in both healthy and GH-deficient adults (6).
GH treatment normalizes or reduces body fat and increases muscle area in GHD patients (7, 8, 9). However, the relation to expression of the GH receptor in both adipose tissue and muscle tissue has not been studied to date.
We studied the relationship among the expression of two different GH receptor types, levels of circulating GHBP, and body composition during GH treatment in GH-deficient adult patients. The aim of the present study was to investigate gene expression of the full-length and the truncated receptor in sc adipose tissue and skeletal muscle before and after GH replacement in GH-deficient adults and to study the relation to GHBP and body composition.
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Subjects and Materials |
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TopAbstractIntroductionSubjects and MaterialsResultsDiscussionReferences |
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Eleven GH-deficient adults participated in the study, from which other data have previously been published (10, 11). The patients were aged (mean ± SEM) 41.5 ± 3.3 yr, and the body mass index (BMI) was 31.6 ± 1.0 kg/m2. All participants were included in the study after giving written informed consent in accordance with Helsinki Declaration II. The patients received GH (Norditropin, Novo Nordisk, Bagsvaerd, Denmark) for 4 months in a dose of 2 IU/m2. Insulin-like growth factor I (IGF-I) levels increased from 117 ± 19 to 337 ± 31 µg/L (P < 0.001). Examinations were performed at baseline and after 4 months.
Tissue biopsies
Adipose tissue biopsies from the abdominal region were obtained by liposuction. The skin was anesthetized with 10 mL lidocaine. Isotonic NaCl was injected into the adipose tissue after the liposuction cannula was inserted, and vacuum was applied using a syringe. The adipose tissue was washed in isotonic NaCl, immediately frozen in liquid nitrogen, and stored at -80 C until analysis. Muscle biopsies were obtained from the lateral vastus muscle with a Bergström cannula under local anesthesia with lidocaine. The biopsies were immediately frozen in liquid nitrogen and stored at -80 C until analysis.
GH receptor mRNA quantitation
RNA was isolated using the TRIzol reagent (Life Technologies, Inc., Grand Island, NY). RT and amplification were performed with 25 ng total mRNA using AmpliTaq Gold DNA polymerase and hexamer as described by the manufacturer (GeneAmp PCR kit, Perkin-Elmer Corp./Cetus, Norwalk, CT). The GHR and GHRtr primers used in the PCR spanned complementary DNA products of 158 and 132 bp, respectively. The sense primer sequences were as follows: 5'-ATTTTCTAAACAGCAAAGGA-3' and 5'- ATTTTCTAAACAGCAAAGTT-3' for GHR and GHRtr, respectively. The reverse primer was the same in the PCR reactions: 5'-CACTGTGGAATTCGGGTTTA-3' (1). ß-Actin mRNA was amplified as a housekeeper marker, and a semiquantitative multiplex PCR method called primer dropping was used to monitor mRNA expression (12). Semiquantitative multiplex PCR estimates the amount of mRNA relative to a known housekeeping gene (ß-actin) working as an internal control of sample variability. We measured the density of the ß-actin bands in adipose and muscle tissues before and after 4 months of GH treatment and found no change in gene expression in muscle tissue (59.6 ± 4.6 vs. 60.9 ± 4.1; P = 0.8) or adipose tissue (49.5 ± 5.7 vs. 53.7 ± 0.5; P = 0.5).
Initial experiments were performed for each set of primers to
determine the number of cycles for exponential amplification of
complementary DNA in muscle and adipose tissues (Fig. 1
). The numbers of cycles were almost in
the same range for muscle and adipose tissue for the different primers.
The target mRNAs (GHR and THRtr) were present in much lower amounts
compared with ß-actin, and therefore they had to be run a number
of cycles before the ß-actin primers were added. It was calculated
from the initial experiments that by a pre-run of 11 cycles with the
GHR primers and an additional 23 cycles with the ß-actin primers the
total cycle number for GHR was 34 cycles, and both were then in the
linear amplification. The same calculations were made for GHRtr; the
pre-run was 17, and the total number of amplification cycles was 40. A
similar set-up was used for negative controls, but reverse
transcriptase was omitted, and no PCR products were detected. The PCR
products were loaded onto a 4% NuSieve gel stained with
ethidium bromide and analyzed using the Gel Doc 1000 system suitable
for quantification (Bio-Rad Laboratories, Inc., Richmond,
CA). The coefficient of variation for the primer dropping method was
10.2%.
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The amounts of intraabdominal (visceral) fat, abdominal sc fat, muscle, and fat area of the mid-thigh region were evaluated by computerized tomography with a Somatom Plus-S scanner (Siemens, Erlangen, Germany). The areas scanned comprised 10-mm cross-sectional slices at the middle thigh and at the umbilicus using 120 kV and 330 mA. Body composition was estimated with a dual energy x-ray absorptiometry scanner (Nordland XR36, Nordland Instruments, Fort Atkinson, WI). Body mass index was calculated as the weight/height ratio (2).
Immunoassays
IGF-I was determined using an in-house RIA after extraction of binding proteins (13). GHBP was measured with an in-house immunofunctional immunometric assay (4). In brief, the assay was performed in plates from a GH kit (Wallac, Inc., Turku, Finland), coated with a monoclonal anti-GH antibody. Calibrator/serum was dispensed in duplicate, followed by a GHBP saturating amount of recombinant human GH. Finally, Eu3+-labeled antibody against GHBP (Mab 263, Agen Biomedical, Australia) was added. Incubation was performed for 24 h. After washing, DELFIA enhancement solution (Wallac, Inc.) was added, and fluorometry was performed (model 1232 fluorometer, Wallac, Inc.).
Statistics
Student’s t test was performed to evaluate changes during the treatment period. Pearson correlation analysis was performed to relate variables. Results are expressed as the mean ± SEM. A significance level of 0.05 was used.
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Results |
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TopAbstractIntroductionSubjects and MaterialsResultsDiscussionReferences |
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)Generally, GHR was expressed at a much higher level than GHRtr, as a lower degree of amplification was required for this receptor. The GHR expression relative to ß-actin expression was unaltered in abdominal sc adipose tissue after 4 months of GH treatment (0.80 ± 0.12 vs. 0.67 ± 0.10; P = 0.28). Levels of mRNA for GHRtr increased significantly (0.71 ± 0.20 vs. 1.48 ± 0.32; P = 0.02). In skeletal muscle GHR was expressed at a higher level after GH treatment (0.22 ± 0.04 vs. 0.35 ± 0.04; P = 0.004). In contrast, GHRtr expression decreased significantly (0.48 ± 0.05 vs. 0.30 ± 0.04; P = 0.02).
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GHBP levels decreased significantly during 4 months of GH substitution from 1.73 ± 0.18 to 1.41 ± 1.14 nmol/L (P = 0.008). Baseline GHBP correlated to BMI (r = 0.63; P = 0.04), but not significantly to other indexes of adiposity (data not shown). Levels of GHBP were not correlated to changes in mRNA levels of GHR or GHRtr or to changes in body composition (data not shown).
Body composition
Intraabdominal fat estimated by computerized tomography decreased from 175 ± 18 to 132 ± 11 cm2 (P = 0.008). Abdominal sc fat decreased nonsignificantly from 330 ± 31 to 311 ± 85 cm2 (P = 0.31). Neither the muscle area nor the area of sc fat of the right femur changed after GH treatment [muscle, 160 ± 9 to 157 ± 10 cm2 (P = 0.63); fat, 85 ± 15 to 84 ± 12 cm2 (P = 0.97)]. Total body fat, estimated by dual energy x-ray absortiometry, decreased significantly from 33.1 ± 2.3 to 29.0 ± 2.2 kg (P < 0.001). The BMI tended to decrease (31.6 ± 1.0 to 31.0 ± 1.0 kg/m2; P = 0.07).
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Discussion |
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Three isoforms of the human GH receptor have been described. One is the full-length receptor encoded by exons 2–10 of the GHR gene. Another form originates from an alternatively spliced mRNA lacking exon 3 (GHRd3). This form is lacking 22 amino acids in the extracellular domain and has lower affinity for GH binding (14). A third form originates from an alternatively spliced mRNA lacking 26 nucleotides in exon 9, which leads to a stop codon at position 280, thereby truncating 97.5% of the intracellular domain of the receptor (GHRtr) (1). This receptor has been demonstrated to possess an increased ability to generate soluble GHBP in human tissues (15). At present, most studies have only described expression of the full-length receptor. Expression of GHR mRNA levels in abdominal sc adipose tissue in GHD patients has previously been studied by Kamel et al. (16), who found that GHR mRNA levels increase after 2–4 months of GH therapy in five adult GHD patients and in five children with Prader-Willi syndrome compared with pretreatment levels. Our results are in agreement with this report with respect to the truncated receptor only, as we found full-length receptor expression to be unchanged. In a recently published study Shuto et al. found reduced GHR mRNA expression in the liver in combination with undetectable levels of GHBP in a severely malnourished patient (17), indicating that GHBP reflected GHR status. We found no evidence that circulating GHBP directly reflects GHR or GHRtr mRNA levels in skeletal muscle or adipose tissue before or after GH substitution therapy. Furthermore, changes in GHBP levels during GH treatment did not parallel changes in mRNA levels of the two receptors in either adipose tissue or skeletal muscle. Our findings are also in contrast to a previous observation that total GHR mRNA in skeletal muscle decreases in parallel with circulating levels of GHBP in patients undergoing abdominal surgery (18). In that study mRNA levels for the GH receptor were not studied in adipose tissue, and our data suggest that opposite changes in adipose tissue and skeletal muscle may occur after changes in GH status. We have no precise explanation for these contrasting findings. However, the use of different methods, the low numbers of subjects studied, and the inhomogeneity of the groups compared may explain some of the variable findings. Any relationship between GHR expression and GHBP may depend on type of tissue, GH status, and several unknown factors.
It is not clear whether the main function of GHBP takes place in plasma by dampening the fluctuation of free GH during pulses or prolonging the half-life (19) or whether the GHBP’s main action occurs locally at the membrane surface by inhibiting GH binding to receptors or enhancing GH effect by prolonging the local half-life (20, 21). If the major function is local, studies of the relationship between gene expression in different tissues of GHR and plasma GHBP may not be that relevant. More thorough studies of membrane-bound GHRs and GHBP may help clarify these aspects.
We previously found a strong correlation between specific indexes of abdominal adipose tissue and levels of GHBP in both healthy and GH-deficient adults. In the present study we only found a positive correlation between GHBP and BMI at baseline. The relatively low number of patients is likely to explain this finding. In line with our previous study (5) we found that GHBP levels declined after GH treatment.
The truncated GH receptor has been demonstrated to inhibit GH action in in vitro systems (15).We observed an increase in GHRtr in adipose tissue after GH treatment, with unaltered expression of GHR. Supposing that the GHRtr also inhibits GH action in vivo, it may be suggested from this observation that that the lipolytic effect of GH is decreased after 4 months of GH treatment compared with that under baseline conditions, because of an up-regulation of the truncated receptor. In contrast, the GH effect in muscle tissue seems to be cumulative and maintained after 4 months, as both an increase in GHR and a decrease in GHRtr were seen. This is in accordance with clinical observations where repetitive measurements of changes in adipose tissue produced by GH treatment have shown that the marked reduction in adipose tissue declines after approximately 6 months (7). We did not find any changes in thigh muscle area in the present study, whereas this has been demonstrated in several studies with prolonged GH treatment (8, 9). We conclude that GH treatment influences mRNA expression of both GHR and GHRtr in adipose tissue and skeletal muscle. mRNA expression of the receptors does not seem to be directly related to changes in body composition or GHBP levels. However, the findings suggest that GH-induced changes in body composition can be explained at the GH receptor level. The significance of the present findings, however, needs further investigation, focusing on membrane-bound GHRs, local production of GHBP, and their relation to the effects of GH and circulating GHBP.
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Acknowledgments |
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Footnotes |
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Received March 13, 2000.
Revised July 18, 2000.
Revised October 24, 2000.
Accepted October 30, 2000.
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