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Original Studies |
Research Center for Endocrinology and Metabolism (R.B., R.W., M.H., B.C., L.M.S.C.); the Department of Medicine, International Pediatric Growth Research Center (R.B.); and the Department of Pediatrics, University of Goteborg, Goteborg; and the Clinical Research Laboratory, Department of Surgery, St. Goran Hospital, Karolinska Institute (F.H.), Stockholm, Sweden
Address all correspondence and requests for reprints to: Dr. Ragnar Bjarnason, International Pediatric Growth Research Center, Department of Pediatrics, Sahlgren’s University Hospital/East, S-416 85 Goteborg, Sweden. E-mail: ragnar.bjarnason{at}pediat.gu.se
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Abstract |
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Metabolically healthy patients, without significant preoperative weight loss, who were undergoing elective abdominal surgery were included in the study. Five patients (one woman and four men) were treated with daily injections of GH (0.3 IU/kg·day) in addition to being given total parenteral nutrition including glutamine (0.28 g/kg·day). The control group consisted of eight patients (three women and five men), who were given glutamine-enriched total parenteral nutrition but no GH. A muscle biopsy was taken from the lateral portion of the quadriceps femoris muscle preoperatively (day 0) after induction of anesthesia. A second biopsy was taken under local anesthesia on postoperative day 3. Total ribonucleic acid (RNA) was extracted from the muscle biopsies, and IGF-I messenger RNA (mRNA) and GHR mRNA were measured by competitive quantitative RT-PCR assays. IGF-I mRNA and GHR mRNA levels were related to the expression of a housekeeping gene (cyclophilin). In the control group, IGF-I mRNA levels decreased from 1505 ± 265 (mean ± SEM) transcripts/cpm cyclophilin on day 0 to 828 ± 172 on day 3 (P < 0.05). In contrast, IGF-I mRNA levels did not change in the GH-treated group (1188 ± 400 transcripts/cpm cyclophilin on day 0 vs. 1089 ± 342 transcripts/cpm cyclophilin on day 3). No statistically significant changes were seen in GHR expression.
We conclude that administration of GH prevents the reduction in IGF-I gene expression in skeletal muscle after abdominal surgery.
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Introduction |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Acquired GH resistance is seen in many different disease states (for review, see Ref.3). The protein wasting seen in catabolic states is a well recognized clinical problem, for example in patients undergoing major surgery. When prolonged, protein wasting increases the risk of complications associated with immunosuppression and muscle weakness (3). Patients undergoing elective abdominal surgery are suitable for studies of acquired GH resistance because the onset of the GH resistance is abrupt and defined. GH therapy has been found to improve nitrogen balance in this group of patients (4), but the mechanisms behind this effect are unknown. We have recently developed a quantitative RT-PCR (Q-RT-PCR) assay (5) that enables us to measure changes in GH receptor (GHR) gene expression in tissue samples obtained by needle biopsies. We now report a new Q-RT-PCR assay for measurement of IGF-I messenger ribonucleic acid (mRNA) in human tissues. In the present study this new assay has been used to determine the effect of GH treatment on IGF-I gene expression in skeletal muscle after major surgery.
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Subjects and Methods |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Patients without demonstrable systemic illness, scheduled for
elective colorectal surgery, were included in the study. The
characteristics of the patients and the surgical procedures are shown
in Table 1
. The patients were given
standard postoperative parenteral nutrition containing 0.15 g
nitrogen/kg·day and including 0.28 g glutamine/kg·day. Energy
was provided as equal amounts of glucose and fat (10% glucose and
20% Intralipid; Pharmacia & Upjohn, Stockholm, Sweden) given at 1.2
times the calculated caloric need according to the Harris-Benedict
formula. During the day of operation, 75% of this nutritional regimen
was given. One group of patients (n = 5) was given daily sc
injections of recombinant human GH (0.3 IU/kg·day; Genotropin,
Pharmacia & Upjohn), whereas the other group served as controls (n
= 8). The study was randomized, but not blinded.
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The study was approved by the ethical committees of Goteborg University (Goteborg, Sweden) and the Karolinska Institute (Stockholm, Sweden). The procedure of the study, possible discomfort, and risks involved were explained to the patients, and their informed consent was obtained before participation in the study.
Q-RT-PCR for IGF-I gene expression
Primers. The primers were designed to amplify a fragment of
the IGF-I complementary DNA (cDNA) that is present in all splice
variants of the IGF-I mRNA (Fig. 1a).
Primers were purchased from KEBO Lab (Spanga, Sweden), except for the
biotinylated primers, which were purchased from Scandinavian Gene
Synthesis AB (Koping, Sweden). The sequences of the primers are listed
in Table 2, and their locations are shown
in Fig. 1b.
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cDNA synthesis and PCR. cDNA was generated from total RNA in 1 x reverse transcriptase buffer [50 mmol/L Tris-HCl (pH 8.3, at 42 C), 5 mmol KCl/L, 1 mmol MgCl2/L, 10 mmol dithiothreitol/L, and 0.05 mmol spermidine/L; Promega Corp., Madison, WI], 7 U AMV reverse transcriptase (Promega), 20 U RNasin (Promega), 1.5 mmol deoxy-NTP/L (Boehringer Mannheim), and 0.5 µg random hexamers (Boehringer Mannheim) in a final volume of 20 µL. Annealing was performed at 22 C for 5 min, followed by extension at 42 C for 50 min; the reaction was terminated at 70 C for 5 min. The PCR was carried out in 1 x PCR buffer [10 mmol Tris-HCl/L (pH 8.3, at 20 C), 50 mmol KCl/L, and 1.5 mmol MgCl2/L; Boehringer Mannheim], using 2.5 U Taq polymerase (Boehringer Mannheim), cDNA solution (20 µL), primer Bio-IGF (15 pmol), and IGF BamHI (50 pmol) in a final volume of 100 µL. All PCR reactions were performed using the GeneAmp PCR system 9600 (Perkin-Elmer/Cetus, Norwalk, CT) programmed for 30 cycles (15-s denaturation at 94 C, 15-s annealing at 57 C, and 30-s elongation at 72 C). Negative controls were always included during cDNA synthesis and PCR to confirm that there was no contamination.
Assay procedure
To minimize the risk of PCR contamination, specific steps of the
assay were carried out in separate rooms for RNA preparation, pre-PCR,
PCR, and post-PCR. The assay procedure is outlined in Fig. 2. Samples of total RNA extracted from
human tissues or synthetic IGF wt RNA (for the standard curve;
0.0156–1.0 x 106 transcripts/reaction) were mixed
with 105 molecules of synthetic IGF mut RNA. In addition,
samples containing only 105 transcripts of mutated RNA were
included as blanks. The samples were reverse transcribed into cDNA, and
the PCR was carried out as described above, using primers Bio-IGF and
IGF BamHI. For immobilization and purification of the PCR
products, the PCR solution (20 µL) was mixed with 40 µL PBS buffer
[0.14 mol NaCl/L, 0.01 mol sodium phosphate buffer/L (pH 7.4), and
0.1% Tween-20] and dispensed into streptavidin-coated microtiter
plates (Streptavidin Covalent Strips, Wallac Oy, Turku, Finland) that
were prewashed four times at room temperature with TENT buffer [40
mmol Tris-HCl/L (pH 8.8), 1 mmol ethylenediamine tetraacetate/L, 50
mmol NaCl/L, and 0.1% Tween-20] in an automated microtiter washer
(Scanwasher, Skantron Instruments, Lier, Norway). The plates were
sealed and incubated for 1.5 h with gentle agitation at 37 C and
subsequently washed four times with TENT buffer. To obtain
single-stranded DNA, the immobilized PCR products were denatured with
100 µL 50 mmol NaOH/L plus 150 mmol NaCl/L twice for 5 min each time
at room temperature, followed by four washes with TENT buffer. The
ratio between the IGF wt and IGF mut sequences was determined by two
separate minisequence reactions, in which the primer IGF seq,
complementary to the sequence immediately 3' to the variable
nucleotide, was extended with a radiolabeled nucleotide, either C
for the IGF wt or G for the IGF mut sequence. The sequence reaction was
carried out at 54 C for 10 min in PCR buffer [10 mmol Tris-HCl/L (pH
9.0 at 25 C), 50 mmol KCl/L, 1.5 mmol MgCl2/L, and 1%
Triton X-100; Promega] containing 0.2 U Taq polymerase
(Promega), the primer IGF seq (0.2 µmol/L), and either
[3H]deoxy-GTP (TRK 625, Amersham International, Little
Chalfont, UK) or [3H]deoxy-CTP (TRK 627, Amersham
International) in a total volume of 50 µL. The microtiter plates were
washed four times with TENT buffer and counted in a microliquid
scintillation counter (Wallac 1450 MicroBeta Plus, Wallac, Oy,
Finland).
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) for
27 cycles (15-s denaturation at 94 C, 15-s annealing at 57 C, and 30-s
elongation at 72 C). [3H]Deoxy-TTP (TRK 933, Amersham
International), 0.8 µmol dideoxy-GTP/L (Boehringer Mannheim), and 0.4
µmol primer CP-seq/L (Table 1) were used in the cyclophilin
minisequence reaction. Measurements of GHR mRNA
GHR mRNA was measured by a Q-RT-PCR-based assay similar to the assay for IGF-I mRNA described above (5).
GH-binding protein (GHBP) assay
Total GHBP was measured by a ligand-mediated immunofunctional assay, as previously described (10). The detection range of the ligand-mediated immunofunctional assay was 15.6–1000 pmol/L. The intraassay coefficient of variation was 7.3%. All samples were analyzed in the same assay.
IGF-I assay
Plasma concentrations of IGF-I were measured by RIA (Nichols Institute Diagnostics, San Juan Capistrano, CA). IGF-I was separated from binding proteins using an acid-ethanol and alkaline precipitation step. At 352 µg/L, the intraassay coefficient of variation was 4.8%, and the interassay coefficient of variation was 3.5%.
IGFBP-3 assay
Plasma levels of IGFBP-3 were measured by RIA (Nichols Institute Diagnostics). At 3.4 mg/L, the intraassay coefficient of variation was 4.2%, and the interassay coefficient of variation was 2.4%.
Statistical analysis
For evaluation of changes before (day 0) and after surgery (day 3), a Wilcoxon signed rank test was used. Changes were considered significant if P < 0.05. Data are presented as the mean ± SEM.
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Results |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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The assay reproducibility was determined by measurements of IGF-I mRNA in replicates of total RNA (50 ng) from human liver. The intraassay coefficient of variation varied between 11.3% (n = 5) and 13.5% (n = 8). IGF-I mRNA was measured in serial dilutions of total RNA extracted from human liver. In samples containing 25, 50, and 100 ng total RNA from the same liver sample, 2434, 2471, and 2432 IGF-I mRNA molecules/ng total RNA were detected, indicating that the assay is linear. The detection limit for the assay was 1.6 x 104 molecules/reaction, as defined by Rodbard et al. (11) to be the equivalent to the concentration corresponding to the mean absorbance of zero plus twice the SD.
IGF-I and GHR mRNA abundance in skeletal muscle biopsies
The IGF-I mRNA levels were measured in skeletal muscle before and
3 days after abdominal surgery in five patients given total parenteral
nutrition (TPN), glutamine, and GH and in eight patients given TPN and
glutamine only (control group). In the control group, IGF-I mRNA levels
decreased after surgery (1505 ± 265 vs. 828 ±
172 transcripts/cpm cyclophilin; P < 0.05; Fig. 3a). In contrast, IGF-I expression did
not change after surgery in the GH-treated group (1188 ± 400
vs. 1089 ± 342 transcripts/cpm cyclophilin;
P = 0.81; Fig. 3b). There were no statistically
significant changes in GHR gene expression in the two groups [199
± 34.1 vs. 184 ± 49.1 transcripts/cpm cyclophilin
(Fig 3c) and 125 ± 19.9 vs. 230 ± 64.0
transcripts/cpm cyclophilin (Fig 3d) for the controls and the
GH-treated subjects, respectively].
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Plasma levels of IGF-I and IGFBP-3 were measured before surgery
and on days 1, 2, and 3 after surgery in all patients. For statistical
analysis, the levels on day 0 were compared with the levels on day 3.
In the control group, plasma IGF-I levels were unchanged (128 ±
26.2 vs. 140 ± 30.8 µg/L; Fig. 4a), whereas there was a significant
decrease in plasma IGFBP-3 (2.5 ± 0.27 vs. 2.0 ±
0.22 mg/L; P < 0.05; Fig. 4c). In the GH-treated group
there was a nonsignificant (P = 0.063) increase in
plasma levels of both IGF-I (139 ± 33.7 vs. 381
± 60.0 µg/L; Fig. 4b) and IGFBP-3 (2.0 ± 0.28 vs.
2.8 ± 0.34 mg/L; Fig. 4d). Plasma GHBP levels decreased from day
0 to day 3 in all patients for whom samples were available for
analysis; unfortunately, only three patients in the GH-treated group
and seven patients in the control group were available for analysis
(data not shown).
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Discussion |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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In this study we examined the effects of GH administration on expression of the GHR gene and the IGF-I gene in patients undergoing abdominal surgery. We demonstrated that although IGF-I levels in plasma were unchanged after abdominal surgery, there was a significant decrease in IGF-I mRNA levels in skeletal muscle 3 days after surgery. These changes were reversed by once daily sc injection of GH.
The IGF-I level in plasma/serum has been proposed as a good indicator of GH activity. However, circulating IGF-I, most of which is believed to originate from the liver, may not reflect the level of IGF-I produced in peripheral target tissues in response to GH. Furthermore, serum concentrations of IGF-I are also influenced by nutrition and body composition. This is a particular problem in adults with GH deficiency, in whom there is a large overlap between patients and controls (15) despite great abnormalities in body composition as a result of GH deficiency (16). In the present study, IGF-I levels in plasma were unchanged in the control group, whereas IGF-I gene expression in skeletal muscle decreased significantly. GH treatment increased IGF-I levels in plasma, whereas IGF-I expression was unchanged in skeletal muscle. The differential regulation of plasma IGF-I and IGF-I mRNA in skeletal muscle together with the assumption that the liver is the major producer of plasma IGF-I suggest that the regulation of IGF-I gene expression is different in skeletal muscle and liver. This emphasizes the potential importance of this new Q-RT-PCR for IGF-I mRNA in studying the different effects of GH in various human tissues and organs. In acquired GH resistance and in some short children, the effects of GH are less than expected, and we believe that measurements of IGF-I and GHR mRNA expression in different tissues might help in the understanding of some of the mechanisms involved.
There was no change in GHR mRNA gene expression after surgery in the control group, in contrast to our previous findings (5). GHBP levels decreased in all patients for whom samples were available for analysis. In our previous study we also demonstrated that both GHR mRNA in skeletal muscle and plasma levels of GHBP fell in parallel after abdominal surgery (5). However, the present protocol is different from that used previously, in that all patients received glutamine in the TPN. This might have affected GHR gene expression, although further studies are necessary to confirm this hypothesis. GH appeared to have a minor, if any, effect on GHR expression in skeletal muscle. This is consistent with the results from previous studies, in which minor changes in GHBP levels occurred after the start of GH treatment in both children (17) and adults (18) with GHD. However, it should be noted that adults with GHD are a much more homogeneous group than children with GHD, in that they have very low levels of GH secretion, if any. The fact that there was no dramatic change in GHBP in adults with GHD further strengthens the argument that GH has little or no role in the regulation of GHBP.
PCR techniques have been used to quantify gene expression in different tissues. The technique is so sensitive that extreme care must be taken to prevent contamination of the sample by previously amplified DNA material. Another problem is that when comparing the abundance of different mRNAs, the efficacy of amplification can vary, which applies both to the RT step and the PCR amplification. By using an internal standard, it is possible to compensate for differences in the efficacy of the cDNA synthesis and the PCR reaction, which could otherwise be a source of considerable intersample variation in RT-PCR assays. The advantage of using an internal standard that differs from the wt mRNA by only one base is that the same primer pair can be used for amplification of both standard and sample. Furthermore, as the fragments differ only by one base, the efficacy of the amplification is similar. Both of these factors are important in excluding selective amplification of one fragment. The two fragments compete for amplification in the PCR reaction, and the ratio between the two is relative to the amount of RNA in the sample, independently of whether the PCR reaction is in the exponential or the plateau phase. As the concentration of the mutated fragment is known, the amount of IGF-I wt mRNA in the sample can be calculated from a standard curve.
In conclusion, we have demonstrated that although plasma IGF-I levels were unchanged after major surgery, there was a significant decrease in IGF-I gene expression in skeletal muscle. GH treatment prevented these changes in IGF-I gene expression and induced a clear trend for an increase in IGF-I and IGFBP-3 levels. The new Q-RT-PCR technique, with its high sensitivity, makes studies possible on small biopsy samples. It, therefore, opens new possibilities for performing dynamic studies on the action of GH and the sensitivities of various tissues to GH.
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Acknowledgments |
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Footnotes |
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Received June 2, 1997.
Revised January 21, 1998.
Accepted January 29, 1998.
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References |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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