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Experimental Studies |
Research Center for Endocrinology and Metabolism, Department of Internal Medicine, Sahlgrenska Hospital (M.H., R.B.W., R.B., B.C., L.M.S.C.), and the Department of Pediatrics, East Hospital (R.B.), Goteborg University, Goteborg; and the Department of Surgery, St. Goran Hospital (F.H., I.W.), and the Anesthesiological Metabolism Unit, Clinical Research Center, Department of Anesthesiology and Intensive Care, Huddinge University Hospital (J.W.), Karolinska Institute, Stockholm, Sweden
Address all correspondence and requests for reprints to: Dr. L. M. S. Carlsson, Research Center for Endocrinology and Metabolism, Department of Internal Medicine, Sahlgrenska Hospital, Goteborg University, S-413 45 Goteborg, Sweden. E-mail: lena{at}ss.gu.se
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Introduction |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Subjects and Methods |
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The study was approved by the ethical committee of Goteborg University and Karolinska Institute. Cultured IM-9 cells and tissues obtained during surgery were rapidly frozen in liquid nitrogen and stored at -80 C. Total RNA was isolated from the biopsies using guanidium thiocyanate-phenol-chloroform extraction, essentially as described by Chomczynski and Sacchi (17). Metabolically healthy patients (two women and seven men; mean age ± SEM, 63 ± 4 yr), under 80 yr of age without weight loss, undergoing elective abdominal surgery were included in the study. The patients underwent colonic resection (n = 6), anterior resection of the rectum (n = 1), rectal amputation (n = 1), or hemihepatectomy (n = 1). Preoperatively, after induction of anesthesia a muscle biopsy was taken from the lateral portion of the quadriceps femoris muscle using the percutaneous needle biopsy technique. Muscle tissue of about 30–40 mg wet weight (ww) was used for determination of GHR mRNA and amino acid determination. The ww was determined immediately, and the muscle sample was then frozen in liquid nitrogen and stored at -80 C until analysis. A second muscle biopsy was taken on the third postoperative day under local anesthesia confined to the skin and fascia only. At the time of biopsy, blood samples were drawn from the antecubital vein for the determination of insulin-like growth factor I (IGF-I), IGF-binding protein-3 (IGFBP-3), and GH-binding protein (GHBP). The patients were given a standardized glucose infusion (3.0 g glucose/kg BW·day) postoperatively.
Primers
Primers were purchased from Scandinavian Gene Synthesis (Koping,
Sweden). Localization of the GHR primers is shown in Fig. 1
, and the sequences of all primers are listed in Table 1.
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A 377-bp fragment of the human GHR complementary DNA (cDNA), extending from nucleotides 201–577 (1), was amplified by RT-PCR, using primers GHR3 and GHR4, and subcloned into pGEM-T (Promega, Madison, WI), generating pGHR34. The identity of the subcloned fragment was verified by DNA sequencing using the PRISM Sequenase Terminator Double Stranded DNA Sequencing Kit (Applied Biosystems, Foster City, CA) and an Applied Biosystems 373A automatic sequencer. Two point mutations were introduced at nucleotides 335 (G to C) and 333 (C to A) using PCR-based mutagenesis (18), introducing a BglII site and destroying a PvuII site. Two overlapping fragments were produced with pGHR34 as template in two separate PCR reactions using primers GHR3 and GHR4M, and GHR3M and GHR4, respectively. A mixture of the two amplified fragments was used as the template for amplification with primers GHR3 and GHR4, regenerating a 377-bp GHR cDNA fragment with the desired mutations. The mutated GHR fragment was subcloned into pGEM-T, generating pGHR34M, and the mutations were verified by DNA sequencing. RNA corresponding to the wild-type GHR standard (GHR-wt) and the mutated internal standard (GHR-mut) was obtained by in vitro transcription with SP6 RNA polymerase (Promega) from NcoI (Promega)-linearized pGHR34 and pGHR34M, respectively. In each case, a single band of complementary RNA of the expected size was observed on an ethidium bromide-stained 1.5% agarose gel. In later experiments, a strategy including purification of polyadenylated [poly(A)] transcripts was applied to ensure that full-length transcripts were produced. To obtain in vitro transcription products with poly(A) tails, restriction fragments from pGHR34 and pGHR34M, respectively, were subcloned into the pSP64 poly(A) vector (Promega), generating pGHR34A+ and pGHR34MA+. Synthetic RNA standards produced from EcoRI (Promega)-linearized pGHR34A+ and pGHR34MA+ were purified with PolyATract mRNA Isolation Systems (Promega).
cDNA synthesis and PCR
First strand cDNA was generated from RNA in 1 x reverse transcriptase buffer [50 mmol/L Tris-HCl (pH 8.3; 42 C), 5 mmol/L KCl, 1 mmol/L MgCl2, 10 mmol/L dithiothreitol, and 0.05 mmol/L spermidine; Promega] with 7 U AMV reverse transcriptase (Promega), 20 U RNAsin (Promega), 1.5 mmol/L deoxy (d)-NTP (Promega or Boehringer Mannheim, Mannheim, Germany), and 0.5 µg random hexamers (Promega) in a final volume of 20 µL. After annealing at 22 C for 5 min, the reaction was carried out at 42 C for 50 min and terminated at 70 C for 5 min. cDNA was used as the template for PCR in 1 x PCR buffer [10 mmol/L Tris-HCl (pH 8.3; 20 C), 50 mmol/L KCl, and 1.5 mmol/L MgCl2; Boehringer] with 0.3 mmol/L dNTP (Promega), 3 U Taq Polymerase (Boehringer), primer B-GHR3 (15 pmol), and primer GHR5 (50 pmol) in a final volume of 100 µL. PCR was performed using GeneAmpPCR system 9600 (Perkin-Elmer/Cetus, Norwalk, CT) 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 both cDNA synthesis and PCR to confirm that there was no contamination.
Assay procedure
To avoid contamination, the Q-RT-PCR assay was carried out in
four distinct laboratory areas; sample preparation area, pre-PCR, PCR,
and post-PCR. The assay procedure is outlined in Fig. 2.
Samples, consisting of total RNA extracted from human tissues or GHR-wt
RNA (for construction of the standard curve), were mixed with 1 x
105 molecules of the internal standard, GHR-mut RNA. The
RNA mixture was reverse transcribed into cDNA, and PCR was carried out
as described above, using primers GHR5 and B-GHR3. To immobilize and
purify single stranded PCR products, aliquots (10 µL) of the PCR
reactions were mixed with 50 µL PBS [0.14 mol/L NaCl, 0.01 mol/L
sodium phosphate buffer (pH 7.4), and 0.1% Tween-20] and dispensed
into streptavidin-coated microtiter plates (Streptavidin Covalent
Strips, Wallac Oy, Turku, Finland). Plates were sealed, incubated at 37
C for 1.5 h with gentle agitation, and washed four times with TENT
buffer [40 mmol/L Tris-HCl (pH 8.8), 1 mmol/L ethylenediamine
tetraacetate, 50 mmol/L NaCl, and 0.1% Tween-20] at room temperature.
The immobilized PCR products were denatured by treatment with 100 µL
50 mmol/L NaOH-150 mmol/L NaCl for 5 min at room temperature. The
denaturation step was repeated, and the plates were washed four times
with TENT buffer. The ratio between GHR-wt and GHR-mut sequences was
determined by two separate minisequence reactions, in which the primer
GHR-SEQ, complementary to the sequence immediately 3' of the variable
nucleotide, was extended with a radiolabeled nucleotide complementary
to either the GHR-wt or the GHR-mut sequence. This reaction was carried
out at 55 C for 10 min in PCR buffer [10 mmol/L Tris-HCl (pH 9.0; 25
C), 50 mmol/L KCl, 1.5 mmol/L MgCl2, and 1% Triton X-100;
Promega] containing 2 U Taq-polymerase (Promega), the
primer GHR-SEQ (0.2 µmol/L), either [3H]dGTP (64
Ci/mmol; TRK 625, Amersham International, Little Chalfont, UK) or
[3H]dCTP (31 Ci/mmol; TRK 627, Amersham; 0.2 µmol/L),
and dideoxy-ATP (Boehringer Mannheim; 0.8 µmol/L) 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
Oy).
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Total RNA was isolated from skeletal muscle from nine patients
undergoing abdominal surgery as described in Subjects and
samples above. GHR mRNA levels were measured as described above
with minor modifications. The abundance of GHR transcripts was related
to the expression of a housekeeping gene, cyclophilin (19), and GHR
mRNA levels were expressed as GHR transcripts per cpm cyclophilin.
RT-PCR of cyclophilin was carried out using primers B-CP1 and CP2
(Table 1
) for 26 cycles. [3H]dTTP (116 Ci/mmol; TRK 933,
Amersham), dideoxy-GTP (Boehringer Mannheim), and the primer CP-SEQ
(Table 1) were used in the cyclophilin minisequencing reaction.
Immunoassays
GHBP. Plasma levels of GHBP were measured by ligand mediated immunofunctional assay (LIFA) as previously described (20). All samples were measured in the same assay, and the intraassay coefficient of variation was 7%. The reagents were kindly provided by Genentech (South San Francisco, CA).
IGF-I. Plasma levels of IGF-I were measured by RIA (Nichols Institute Diagnostics, San Juan Capistrano, CA). IGF-I was separated from the binding protein using an acid-ethanol and alkaline precipitation step. The intraassay coefficient of variation was 5%.
IGFBP-3. Plasma levels of IGFBP-3 were measured by RIA (Nichols Institute Diagnostics). The intraassay coefficient of variation was 4%.
Amino acid determination
The free amino acids in skeletal muscle were determined with special emphasis on glutamine and branched chain amino acids (21). To denature the proteins, the biopsy specimens were homogenized in 4% sulfosalicylic acid containing nor-leucine as an internal standard. The precipitated proteins were sedimented by centrifugation, and the supernatant was used for determination of free amino acids. The free amino acids in the supernatant were separated by ion exchange chromatography (Alpha Plus, LKB, Bromma, Sweden), derivatized with o-phthaldialdehyde on DC-6 ion exchange resin (Durrum, CA) and lithium citrate buffers, and quantified using a fluorescence detector (21).
Nitrogen balance determination
Urine was collected over 24-h periods. By determination of the nitrogen content of the urine, nitrogen losses could be calculated. The urinary nitrogen content was determined by chemiluminescent nitrogen analyzer (771 C pyroreactor, 720 C nitrogen detector, Antek Instruments, Houston, TX). The external nitrogen losses per 24 h were approximately 1.5 g and were included in the calculation. The cumulative nitrogen balance during the study period was calculated.
Statistical analysis
For evaluation of GHR mRNA, GHBP, IGF-I, IGFBP-3, and amino acids before and after surgery, a paired t test was used. Data are represented as the mean ± SEM.
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Results |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Figure 3 shows three Q-RT-PCR standard curves
obtained by serial dilutions of GHR-wt RNA mixed with a fixed amount
(1 x 105 molecules) of GHR-mut RNA. The RNA samples
were reverse transcribed, amplified, and detected as described in
Subjects and Methods. The assay range for the Q-RT-PCR assay
was 0.125–8 x 105 GHR-wt transcripts/sample. The
lower limit of detection was 0.125 x 105, as defined
by Rodbard et al. (22) to be equivalent to the concentration
corresponding to the mean absorbance of zero plus twice the
SD.
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Replicates of samples containing 25, 50, or 100 ng total RNA
extracted from cultured IM-9 cells were analyzed for the assessment of
intraassay precision (Table 2). The intraassay
coefficients of variation ranged from 6.9–15.9%, and the average
coefficient of variation was 8.7%.
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The linearity of the assay was determined by measurements of GHR
mRNA by the Q-RT-PCR assay in serial dilutions of total RNA (40, 20,
and 10 ng) extracted from human liver. After correction for the
dilution factor, approximately 6.8 x 106 GHR
transcripts/µg total RNA were found in all three samples (Fig. 4), indicating that the assay is linear.
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To compare GHR gene expression in different human tissues, total
RNA was extracted from liver (n = 1), skeletal muscle (n =
3), and adipose tissue (n = 5), and GHR mRNA concentrations were
measured by Q-RT-PCR. The GHR gene expression was highest in liver
(13 x 106 transcripts/µg total RNA), whereas the
expression in skeletal muscle and adipose tissue was approximately 10
and 3 times lower, respectively (Fig. 5). The expression
levels also varied between subjects. GHR gene expression ranged between
2.4–5.2 x 106 transcripts/µg total RNA in adipose
tissue and between 0.4 and 1.9 x 106 transcripts/µg
total RNA in skeletal muscle.
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To determine whether surgical trauma is related to changes in GHR
gene expression, the Q-RT-PCR assay was used to measure GHR mRNA in
skeletal muscle from nine patients undergoing major abdominal surgery
(Fig. 6A). GHR mRNA levels were significantly reduced
from 450 ± 85 transcripts/cpm cyclophilin at baseline to 291
± 48 transcripts/cpm cyclophilin on day 3 after surgery
(P < 0.05). In addition, plasma levels of the
GHR-related GHBP, IGF-I, and IGFBP-3 were measured. GHBP levels were
reduced from 232 ± 40 pmol/L at baseline to 155 ± 31
(P < 0.01), 138 ± 30 (P <
0.01), and 160 ± 33 (P < 0.05) on days 1, 2, and
3 after surgery, respectively (Figs. 6B and 7C). IGF-I
decreased from 163 ± 18 µg/L at baseline to 153 ± 24
(P = NS), 135 ± 21 (P < 0.01),
and 123 ± 24 (P < 0.01) µg/L on days 1, 2, and
3 after surgery, respectively (Fig. 7A). IGFBP-3 decreased from
2.36 ± 0.15 mg/L at baseline to 1.99 ± 27
(P = NS), 1.89 ± 0.23 (P <
0.05), and 1.73 ± 0.21 (P < 0.01) mg/L
respectively on days 1, 2, and 3 after surgery (Fig. 7B).
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) and with an increase in muscle free
branched chain amino acids from 0.35 ± 0.03 mmol/kg ww muscle at
baseline to 0.50 ± 0.03 mmol/kg ww muscle tissue on day 3 after
surgery (P < 0.05; Table 3). The total amino acid
concentration decreased from 20.84 ± 0.97 mmol/kg ww muscle at
baseline to 13.28 ± 1.23 mmol/kg ww muscle on day 3 after surgery
(P < 0.001; Table 3). The cumulated nitrogen balance
during the study period was -27.7 ± 3.4 g.
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Discussion |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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It is difficult to compare the quality and performance of different
Q-RT-PCR assays because the validation is usually not published. In
contrast to bioassays and RIAs, for which strict guidelines for assay
validation are provided in the instructions to authors, there are no
guidelines for how PCR-based assays should be validated. Because of the
acknowledged pitfalls common to PCR, we suggest that validation of
Q-RT-PCR assays should include assessment of assay precision and
linearity. Measurement of GHR mRNA in samples containing different
amounts of total RNA extracted from a human liver demonstrated
parallelism of standard and unknown sample in our assay. Although the
intraassay variability was in the same range as that for many
immunoassays, in our experience the interassay variability was higher
(
17–32% depending on transcript abundance in the sample; data not
shown), suggesting that comparisons should preferably be made within
the same assay. Estimation of the lower limit of detection directly
provides important information for an immunoassay. A certain level of
detection for the Q-RT-PCR assay, expressed as transcripts per sample,
may imply that the assay can be used to measure mRNA levels in some
tissues but not in others, as expression levels often vary between
tissues.
Our Q-RT-PCR assay was developed to allow studies of GHR gene expression in human tissues. The assay is very sensitive and can be used to measure GHR mRNA levels in total RNA isolated from needle biopsies. Biopsies of different human tissues are often taken to aid the diagnosis of various diseases, but unless PCR-based assays are used, the amount of tissue that can be obtained is too small to allow studies of low abundance mRNAs, such as GHR mRNA. Therefore, very little is known about the physiological and pathophysiological regulation of GHR gene expression in human tissues. We used the Q-RT-PCR assay to measure the GHR mRNA levels in three of the major target tissues for GH (liver, skeletal muscle, and adipose tissue) and showed that GHR mRNA was clearly measurable in all samples tested. The GHR mRNA levels that we report are slightly higher than the levels measured by another Q-RT-PCR assay developed by Martini et al. (11). However, the relative difference between GHR gene expression in liver and muscle was similar and also fits well with reports of GHR gene expression in these tissues, measured by other techniques, in the rat (26).
Indirect evidence suggests that many diseases and syndromes are accompanied by reduced sensitivity to GH. Hormone insensitivity may have several causes, including structural defects in the receptor, receptor down-regulation, or postreceptor defects. With the exception of patients with Laron’s syndrome and some children with idiopathic short stature, in whom GH resistance is genetic and caused by mutations in the GHR gene, (27; reviewed in Ref.3), the mechanisms behind the GH resistance are unknown. Structural defects due to deletions or mutations in receptor genes are interesting and important because they provide information about the structure-function relationship of the receptor. Furthermore, structural defects can explain the hormone insensitivity in some patients. However, in most patients, hormone insensitivity is acquired, indicating that a decrease in the abundance or activity of the receptor or postreceptor molecules may be the cause.
GH resistance is found in conditions associated with an increased catabolic rate, such as trauma, sepsis, and surgery (reviewed in Ref.4). Typical changes in the GH-IGF-I axis in such conditions of GH resistance include up-regulated GH secretion, low levels of IGF-I and IGFBP-3, high levels of IGFBP-1, and reduced concentrations of GHBP (4, 14). A reduction in skeletal muscle mass is also a major consequence during catabolic illness. The loss of skeletal muscle mass is accompanied by well described metabolic changes in skeletal muscle, such as a decrease in protein synthesis (28), a reduction of muscle free glutamine, and increases in branched chain amino acids (29). The degree of catabolism can be assessed by the amount of nitrogen lost during the period studied. To determine whether these changes may be explained by changes in GHR gene expression, we used the Q-RT-PCR assay to measure GHR mRNA levels in skeletal muscle from nine subjects undergoing abdominal surgery. In addition, we measured GHBP, which corresponds to the extracellular domain of the GHR and is thought to be derived proteolytically from the cell membrane-bound receptor (1, 2, 30). The patients in the present study showed parallel decreases in GHR gene expression, plasma levels of GHBP, and muscle free glutamine as well as pronounced nitrogen losses. The changes in free glutamine and nitrogen losses indicate a pronounced state of muscle protein catabolism, with the loss of about 1 kg skeletal muscle during 3 days. It has been demonstrated that postoperative muscle protein catabolism can be reduced by treatment with recombinant human GH as an adjuvant to nutrition (31, 32, 33) as well as by the addition of glutamine to the parenteral nutrition (34). The results from the present study indicate that expression of the GHR gene in skeletal muscle is reduced postoperatively. Because skeletal muscle represents the bulk of peripheral tissue, this may explain part of the metabolic events in catabolic illnesses characterized by an increased flow of substrate from peripheral protein stores to more central tissues, such as the splanchnic organs, and immunocompetent tissues (35, 36, 37).
The factor mediating the reduction of GHR gene expression in skeletal
muscle after major surgery is unknown. An interesting group of
candidate molecules is the cytokines. Administration of several
cytokines has been shown to induce catabolism (38, 39), and several
cytokines are produced in response to trauma and infections (40). One
of the cytokines, tumor necrosis factor-
, shows increased expression
in adipose tissue in obesity and has been suggested to cause insulin
resistance in skeletal muscle (41). A similar mechanism may be involved
in the decrease in GHR gene expression in skeletal muscle in patients
with inflammatory processes, for example after major surgery.
Interestingly, cytokines, including interleukin-1ß and tumor necrosis
factor-, inhibit GHR gene expression in rat hepatocytes in
vitro (42).
We conclude that the Q-RT-PCR assay described in this study is sensitive and reliable. Using this assay it was possible to demonstrate reduced GHR gene expression in skeletal muscle in patients after major surgery.
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Acknowledgments |
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Footnotes |
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2 M.H. and R.W. contributed equally to this manuscript and are
considered to be equal first authors.
Received July 12, 1996.
Revised October 14, 1996.
Accepted October 21, 1996.
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: direct role in
obesity-linked insulin resistance. Science. 259:87–91.This article has been cited by other articles:
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