Induction of Growth Hormone Receptor and Insulin-Like Growth Factor-I mRNA in Aorta and Caval Vein During Hemodynamic Challenge
Induction of two-kidney, one clip hypertension (renal hypertension) is characterized by a slow increase in left ventricular tension and aortic wall stress, as opposed to aortocaval fistula or shunt volume overload, which induces a marked and rapid onset of wall stress in the caval vein and right ventricle. In the present study, we applied hemodynamic challenge to study the growth response involving gene expression of insulin-like growth factor-I (IGF-I) and growth hormone receptor (GH-R) mRNA in aorta and caval vein. Volume overload and pressure overload were induced in Wistar rats by means of shunt and renal hypertension, respectively. Systolic pressure was measured before excision of the great vessels, which was performed between 2 and 12 days postoperatively. Aortic and caval vein IGF-I and GH-R mRNA expressions were measured by means of a solution hybridization assay, and the caval vein was analyzed for IGF-I protein by immunohistochemistry. In the volume-distended but not pressurized caval vein in shunt rats, verified by telemetry recordings, there was an eightfold increase in IGF-I and 3.5-fold increase in GH-R mRNA at day 4 versus control. The IGF-I protein appeared to be localized in smooth muscle cells. In the aorta of the renal hypertension group, changes were of a slower onset. At day 7, there was a fourfold increase in IGF-I and fivefold increase of GH-R mRNA expressions versus sham-operated rats. Both the shunt caval vein and renal hypertension aorta showed evidence of a structural adaptation of the growth response. The present study suggests that acute elevation in vascular wall stress is an important triggering factor for overexpression of IGF-I and GH-R mRNA in great vessels. The growth hormone/insulin-like growth factor axis may be an important link in mediating structurally adaptive growth responses in the blood vessel wall.
Adaptive growth of the heart and vessels occurs normally in response to a hemodynamic challenge. This adaptation normalizes the elevated wall stress.1 Cardiac growth results predominantly from myocardial cell hypertrophy in the absence of myocyte cell proliferation. In contrast, vascular growth is shown to be a consequence of different changes in vascular smooth muscle cells such as increased cell size (hypertrophy), increased cell number (hyperplasia), or elevated DNA content per cell without a concomitant increased cell number (polyploidy).2 3 4 5 6 7
A complex interplay between hemodynamic factors—such as transmural pressure, shear stress, flow, pulsatility, systolic wall stress, and circulating vasoactive substances, such as endothelin, Ang II, norepinephrine, and various growth factors—may promote cardiovascular tissue growth.8 9 10 11 A number of studies have indicated an important role for IGF-I on cardiovascular development and tissue growth response.12 13 14 15 IGF-I acts both as a hormone and at the tissue level in an autocrine, paracrine, or intracrine manner.16 Furthermore, GH is the primary regulator of IGF-I synthesis and mRNA content.17 18 Some studies also suggest that tissue growth response depends on GH-R expression.19 20 21 Increased IGF-I expression, at both the mRNA and protein levels, is associated with the development of cardiovascular hypertrophy and hyperplasia during hemodynamic overload, ie, renal hypertension (2K1C), ACF, and aortic constriction, respectively,12 13 14 15 22 23 suggesting that the overexpression of IGF-I mRNA is mediated via an increased systolic wall stress. Whether a similar IGF-I and GH-R gene induction pattern, as occurs in cardiac tissue during hemodynamic challenge, also applies for vascular tissue has yet to be determined, although vascular smooth muscle cells have been shown to express increased IGF-I mRNA levels in relation to increased wall stress, as exemplified in an in vitro system24 and in vivo in obstructed urinary bladder and portal vein.25
Alterations in the circulation induced by experimental manipulations such as 2K1C and ACF promote structural adaptive responses, although the patterns of hemodynamic changes are widely different. An ACF results in a vigorous elevation of volume flow from the arterial to the venous side, leading to widening of the caval vein, which promotes a marked increase in vascular wall stress; or as demonstrated by Dobrin,26 increased circumferential stress initiates medial thickening. In contrast, the arterial system is unloaded with a reduction of afterload for the left ventricle, although the diastolic wall stress is increased. On the other hand, in the 2K1C model, arterial blood pressure increases slowly and persistently during the first 2 to 3 weeks after intervention.27 Comparison of these contrasting experimental models shows that the arterial circulation and left ventricle are less challenged in the early phase of the 2K1C hypertensive model, whereas the venous side and right ventricle receive an immediate and pronounced hemodynamic stimulus in the ACF model.
Currently, little is known about the in vivo involvement of components of the GH/IGF-I axis in vascular tissue during hemodynamic overload, especially with regard to great veins, an issue of particular relevance in bypass grafting surgery. The aim of the present study was to characterize the magnitude and time course of hemodynamic parameters and growth response processes involving the patterns of IGF-I and GH-R mRNA expressions in the caval vein and aorta in the 2K1C and ACF models, respectively. Moreover, the existence of IGF-I protein was visualized by means of immunohistochemistry in the volume-overloaded caval vein.
All rats were obtained from Möllegaard Breeding Center Ltd (Ejby, Denmark) and arrived 1 week before the start of experimentation. They had free access to standard pellet chow and tap water throughout the study. All experiments were approved by the Regional Animal Ethics Committee in Göteborg.
Volume overload was induced during short-lasting anesthesia with barbiturate methohexital sodium (Brietal, Eli Lilly & Co; 75×10−3 g/kg body wt IP) in male normotensive Wistar rats weighing 160 to 200 g by means of a fistula between the aorta and inferior caval vein.28 The abdominal aorta was punctured with an 18-gauge disposable needle and advanced into the inferior caval vein. After the needle was withdrawn, the puncture site was sealed with cyanoacrylate glue. Before the abdomen was closed, the patency of the shunt was confirmed visually by mixing of arterial blood in the caval vein. A sham operation was performed in control rats by merely exposing the aorta and inferior caval vein.
Experiments were performed in ACF (n=8) and sham-operated (n=4) rats 4, 8, and 12 days postoperatively. The degree of shunting was moderate, without signs of cardiac failure, as demonstrated by no significant change in the ratio of liver wet weight to dry weight. To confirm the patency of the shunt, we checked for the presence of a systolic shunt murmur by auscultation. Only ACF rats showing decreased systolic pressure and clear-cut cardiac hypertrophy were included in the study. One rat at day 4, 2 rats at day 8, and 4 rats at day 12 were excluded per the above exclusion criteria.
Radiotelemetric transducers (model TA11PA-C40, Data Sciences Inc) were implanted at least 2 weeks before ACF surgery in separate groups of male Wistar rats. The implantation was made into the abdominal aorta (n=5) or caval vein (n=5) through a midline laparotomy. The tip of the catheter faced upstream in the caval vein and downstream in the aorta, at least 1 cm below the renal artery. The catheter was held in place with a drop of cyanoacrylate glue. The transducer radio signals were collected by a receiver system (RA1010, Data Sciences) placed directly under the rat's cage, and the digital signal was transferred via a consolidation matrix (BCM100, Data Sciences) to a computer-based data-acquisition system (Dataquest IV, version 4, Data Sciences). Arterial and venous pressures were collected on-line at 150 Hz for 15 seconds at 10-minute intervals for each rat. Mean arterial and central venous pressures were monitored by the Dataquest IV software program and stored for later analysis. The shunt was created at the same level as the telemetry catheter tip in the caval vein and aorta, respectively. During the ACF operation, pressure monitoring was stopped for 2 hours.
2K1C Renal Hypertension
During short-lasting anesthesia as described above, 2K1C was induced in normotensive male Wistar rats weighing approximately 180 g by placement of a silver clip (0.18-mm diameter) around the left renal artery. The right kidney was left intact. A sham operation exposing the left renal artery was performed in control rats.
Experiments were performed on 2K1C (n=8) and sham-operated (n=4) rats 2, 4, and 7 days after surgery. Early systolic pressure elevation (≥15% increase) was used as the inclusion criterion.27 Two rats at day 2, 3 rats at day 4, and 3 rats at day 7 were excluded from the study because of normotensive blood pressure.
On the day of experimentation, rats were weighed and had their systolic pressure and heart rate measured by tail plethysmography (Narco BioSystems). After anesthesia, the heart, aorta, and caval vein were excised. The vessels were excised directly above the shunt toward the heart, with no tissue included from below the fistula. The vessels were trimmed of extra tissue and weighed before being snap-frozen in liquid nitrogen and stored at −80°C. RV and LV weights from both groups were measured for determination of the degree of hypertrophy. The vessel weights were also related to DNA content.
Molecular Biology and Immunohistochemical Procedures
The pT7T3 18 U plasmid contains a 560-bp BamHI fragment of the rat GH-R cDNA that encodes the early part of the extracellular domain of the GH-R and thus does not differentiate between the GH-R and GH binding protein. Antisense GH-R 35S-UTP–labeled RNA was synthesized according to the manufacturer's instructions (Promega).
A 153-bp Sma I fragment of a genomic subclone of mouse IGF-I (exon 3 by analogy to human IGF-I) subcloned into a pSP64 plasmid was used as a template for probe synthesis.17 The plasmid was linearized with EcoRI and used as a template for synthesis of 35S-UTP–labeled IGF-I cRNA with SP6 RNA polymerase according to the manufacturer's instructions (Promega).
Hepatocyte Growth Factor Probe
To study the specificity of the induction of vascular gene expression, we determined the levels of hepatocyte growth factor. A 650-bp rat hepatocyte growth factor cDNA probe was labeled with 35S-UTP and synthesized with an in vitro transcription set according to the manufacturer's instructions (Promega) using a 1.4-kb EcoRI insert of pRBC1 in an Sac I plasmid linearized with BglII.
Frozen tissue was homogenized and treated with proteinase K, and total nucleic acids were extracted with phenol/chloroform as previously described.29 A solution hybridization assay was used for quantification of IGF-I and GH-R mRNA.29 The probes were hybridized to total nucleic acid samples at 70°C for 24 hours. After RNase treatment, the precipitated protected hybrids were collected on glass-fiber filters (GF/C, Whatman International Ltd) and counted in a scintillation counter. The signal was compared with a standard curve based on known amounts of IGF-I or GH-R mRNA. The results were related to the DNA content in the total nucleic acid sample.30
The specificity of the probes used in the solution hybridization assay was confirmed by separation of the protected fragments on a gel. Total RNA was prepared according to the modified method of Chomczynski and Sacchi.31 Samples of 25×10−6 to 35×10−6 g were hybridized in a buffer containing 22×10−3 mol/L Tris, 5×10−3 mol/L EDTA, 0.6 mol/L NaCl, 0.075×10−3 g/10−3 L tRNA, 0.75×10−3 mol/L dithiothreitol, 25% formamide, 0.1% (wt/vol) sodium dodecyl sulfate, and 500 000 cpm 35S-labeled probe and were incubated overnight at 70°C.
Single-stranded RNA was digested by addition of 300×10−6 L digestion buffer containing 10×10−3 mol/L Tris-HCl (pH 7.5), 300×10−3 mol/L NaCl, 5×10−3 mol/L EDTA, 40×10−6 g RNase A/10−3 L, and 2×10−6 g RNase T1/10−3 L in a 30-minute incubation at 37°C. After proteinase K digestion, the RNA was extracted with phenol/chloroform and precipitated with 2.5 vol ethanol using 10×10−6 g tRNA as carrier. The pellets were resuspended in loading buffer containing 80% formamide, 15% Ficoll, and 0.25% bromphenol blue. The protected fragments were separated by electrophoresis on 8×10−3 mol/L urea/6% polyacrylamide denaturing gel. As a molecular size marker, an end-labeled ∅ 174/HaeIII DNA marker (Promega) was prepared. The dried gel was exposed on a PhosphoImager screen (Molecular Dynamics Inc) for 3 to 7 days.
Three male Wistar-Kyoto rats underwent ACF surgery, and three sham-operated rats served as controls. Four days after surgery, the rats were euthanized and the caval vein was excised and embedded in OCT compound, rapidly frozen in 2-methylbutane chilled in liquid nitrogen, and stored at −80°C until further use.
Antiserum for rat IGF-Ib prohormone
The antiserum was raised against a 23–amino acid synthetic peptide corresponding to a part of the rat IGF-Ib E-domain. The antiserum has been shown to specifically recognize IGF-Ib in immunoprecipitation experiments.32
Cryostat sections (6×10−6 m) were cut, adhered to microscope glasses, and air-dried followed by fixation in 4% paraformaldehyde in PBS, pH 7.4, for 15 minutes. The sections were blocked with 5% (wt/vol) dried fat-free milk in PBS and quickly rinsed, followed by incubation with the IGF-Ib antisera diluted 1:1000 in 0.5% (wt/vol) dried fat-free milk in PBS at 4°C overnight. The sections were rinsed three times in PBS and incubated for 1 hour at room temperature with anti-rabbit horseradish peroxidase–conjugated Fab fragments (Amersham) diluted 1:50 in 0.5% milk-PBS followed by three additional rinses and development in DAB-chromogen (1×10−3 g/10−3 L DAB and 0.02% H2O2). Two types of control experiments were used: (1) deletion of primary antibody and (2) incubations with unrelated nonimmune rabbit sera diluted 1:1000.
Values are given as mean±SE. Statistical differences between groups were analyzed by one-way and two-way ANOVA followed by the Bonferroni post hoc test. With regard to time course effects, most changes have been assessed statistically considering the entire study period. A value of P<.05 was considered statistically significant.
Table 1⇓ shows data for ACF rats. Systolic pressure was decreased at day 4 (−25%, P<.05) and returned toward normal at the end of the experimental period. With the telemetry technique, mean arterial pressure decreased within the first hour by 20% and by 37% over the next few hours (119±1 to 87±2 mm Hg, P<.05), whereas central venous pressure remained unaltered (6.4±0.4 to 6.0±0.3) during the 10 hours after fistula induction (Fig 1⇓). RV weight was increased by 42% at day 4, 51% at day 8, and 58% at day 12 (P<.05). Similarly, LV weight increased by more than 50% by the end of the experimental period (P<.05). The RV/LV ratio increased some 14% over the experimental period (P<.05). The caval vein and aortic wet weights did not show any statistically significant difference between the intervention and sham groups. The caval vein wet weight related to DNA content (10−3 g/10−6 g DNA) was increased fourfold at day 4 (P<.05), although days 8 and 12 exhibited no statistically significant difference between ACF and sham rats. Aortic wet weight per 10−6 g DNA did not change throughout the study period.
IGF-I mRNA expression in the caval vein showed an eightfold increase at day 4 and a subsequent return to normal expression by day 12 (P<.05, Fig 2A⇓, Table 2⇓). No changes in aortic IGF-I mRNA expression were detected at any of the three time points (Fig 2B⇓, Table 2⇓). Caval vein GH-R mRNA expression was increased by approximately fourfold both 4 and 8 days after fistula induction. GH-R gene expression was normalized at day 12 (P<.05, Fig 3A⇓, Table 2⇓).
Renal Hypertensive Rats
Table 1⇑ also shows data for the 2K1C groups. Systolic pressure increased progressively (P<.05) from days 2 (15%) and 4 (30%) up to day 7 (40%). The LV weight was increased (P<.05) already at day 2 (28%), day 4 (31%), and day 7 (42%), whereas RV weight showed no difference between 2K1C and sham rats. The RV/LV ratio decreased by some 19% over the 7-day experimental period (P<.05). Aortic wet weight showed a statistically significant difference between operated and sham groups during the study course (P<.05), whereas no difference was observed in caval vein wet weight between 2K1C and sham rats. Aortic and caval vein wet weights related to DNA content were similar in 2K1C and sham rats.
Aortic IGF-I mRNA was unchanged 2 days after renal clipping, whereas at 4 and 7 days after clipping, there was a 2.2-fold and 3.4-fold elevation of IGF-I gene expression, respectively (P<.05, Fig 2D⇑). No induction above control level of IGF-I mRNA could be detected in the caval vein (Fig 2C⇑, Table 2⇑). There was a fivefold increase in aortic GH-R mRNA 7 days after induction of renal hypertension versus sham operation (P<.05, Fig 3B⇑, Table 2⇑). No change was found at the two earlier time points.
IGF-Ib immunoreactivity was demonstrated in the caval vein of ACF and sham rats (Fig 4a and 4b⇓⇓). In the caval vessel wall of the ACF rats, IGF-Ib appeared to be increased in cells of the media, possibly in smooth muscle cells, and also in cells in the adventitia. In sections incubated with control nonimmune rabbit serum, only faint background staining was seen (Fig 4c⇓). No staining was observed when the primary antibody was omitted (data not shown).
The caval vein and aorta in the two intervention groups did not show any statistically significant difference over time in hepatocyte growth factor levels, and values were similar to those of the sham group (data not shown).
RNase-protected probe fragments were analyzed on denaturing polyacrylamide gels. For IGF-I (Fig 5⇓, top), the undigested probe was found to be approximately 170 bases long, consisting of a 153-base insert and additional polylinker bases (lane 1). One hundred and twenty bases were protected when the probe was hybridized to RNA from aorta, caval vein, and liver (lanes 6, 7, and 8, respectively), with the smaller size probably reflecting a certain mismatch between mouse probe and rat RNA. A background transcript of approximately 125 bases was also protected (lanes 2 through 8). This mismatch in protected fragments on the gel could be reflected in the results of the solution hybridization assay but would probably cause equally high background values in both the experimental and sham groups. Moreover, for the GH-R (Fig 5⇓, bottom), the undigested probe was found to be 560 bases long, corresponding to the insert (lane 1). Four hundred and ten bases were RNase-protected when the probe was hybridized to RNA from aorta, caval vein, and liver (lanes 6, 7, and 8, respectively). The smaller size of protected fragment reflects the fact that the first part of the probe is not homologous to the rat GH-R mRNA.19
In the present study, we have investigated whether increased systemic pressure overload and volume overload induce IGF-I and GH-R gene expressions in the blood vessels in a manner similar to that found in previous studies in the heart.14 15 The vascular content of IGF-I and GH-R in two different models of hemodynamic overload was analyzed at the mRNA level, and in ACF rats, also at the protein level. The present study suggests that increased vascular loading, along with activation of various neurohormonal systems, stimulates GH-R and IGF-I gene expressions in the vascular wall in 2K1C rats (aorta) and ACF rats (caval vein). In particular, the slow and rapid onset of changes in systolic wall stress may constitute one important factor for the time course and magnitude of gene induction and subsequent structural adaptive growth in the vessel wall. Furthermore, a supporting finding is provided by the increased expression of IGF-I at the protein level, which is likely to occur in vascular smooth muscle cells, as indicated by immunohistochemical staining.
The relationship between hemodynamic stimulation and growth factor gene expression is important when one is comparing the magnitude and rate of onset of a circulatory challenge. However, both the caval vein and aorta show different features regarding structure and function. Thus, when a fistula is created between the abdominal aorta and caval vein, the latter will be immediately exposed to an increase in volume flow and right atrial pressure.33 34 35 36 Surprisingly, radiotelemetry for blood pressure recordings showed no changes in central venous pressure but an immediate and prompt fall in mean arterial pressure and an increase in heart rate after induction of the fistula (Fig 1⇑). This is an intriguing finding, because it means that the venous circulation has the capacity to accommodate rapidly, and to a substantial degree, a large increase in volume flow without increasing pressure. Hence, capacitance vessels must be markedly distended. Moreover, using the ultrasound/Doppler technique,37 we showed a 2.4-fold increase in RV cardiac output 7 days after fistula surgery versus sham rats (280±5 versus 118±2×10−3 L/min, P<.05, unpublished results). Taken together, these observations suggest that the prompt and immediate increase in volume flow to the venous circulation, without any changes in central venous pressure, causes a marked increase in vascular wall stress, hence rapidly stimulating the GH/IGF-I axis locally in the vascular wall.
In 2K1C renal hypertension, in which the aorta is exposed to a more gradual increase in systolic wall stress, a more moderate and late increase in IGF-I mRNA levels occurred. Comparing this finding with that which is observed in the caval vein of ACF rats, it seems reasonable to assume that aortic IGF-I synthesis also depends on an elevation in blood pressure and wall stress. It would then seem that the differences between these experimental models in time course and magnitude, with regard to gene induction, are possibly a consequence of both a relatively smaller increase in aortic wall stress in the 2K1C groups and the large and prompt increase in the caval vein wall stress in the ACF groups.
The lack of overexpression of IGF-I and GH-R mRNAs in the aorta of the ACF groups and in the caval vein of the 2K1C groups was probably due to the unchanged (2K1C) or even reduced (ACF) systolic wall stress. This is in agreement with earlier findings in hearts14 15 in which IGF-I and GH-R gene expression was not increased despite a substantial increase in diastolic wall stress in the left ventricle of ACF animals as well as in the unchallenged right ventricle of 2K1C rats. It should be noted that the left ventricle in ACF animals demonstrates hypertrophy to a similar extent as the right ventricle, indicating that IGF-I is not the only important factor for generating a hypertrophic response, at least in the heart. One can speculate that the elevated diastolic wall stress in the left ventricle of ACF animals is of importance for promoting growth and may trigger the expression of other growth factors.38 In agreement with the present study, Calderone and coworkers39 have shown that various forms of hemodynamic overload are associated with different molecular phenotypes and patterns of peptide growth factor induction.
In the present study, both the ACF caval vein and 2K1C aorta showed evidence for adaptive growth during the first 4 days after surgery. The caval vein in the ACF group showed a fourfold increase in wet weight per DNA content at day 4 compared with the respective sham groups. In contrast, the aorta in the 2K1C groups showed no change in wet weight per DNA content versus sham, whereas aortic wet weight in the 2K1C groups showed a small but significant increase in wet weight. Taken together, the present results show both gene induction and evidence for structural growth, but they do not shed light on the precise nature of the growth response, eg, hypertrophy, hyperplasia, or polyploidy, although the ACF caval vein showed evidence for hypertrophy. Furthermore, it is reasonable to assume that the peak growth response may vary during the first week after induction of pressure and volume load,14 15 depending on the intensity of the hemodynamic stimulus, the biological variance, and the prevailing structure of the vascular wall. However, we cannot rule out the possibility that aortic and caval vein tissues have different, inherent responsiveness when exposed to hemodynamic loading. Accordingly, such a situation could yield a different pattern of gene expression of the components in the GH/IGF-I axis in the two vessel types.
GH and thyroxine have previously been demonstrated to be involved in the development of LV hypertrophy and structural adaptation of the skeletal muscle vascular bed in renal hypertensive, hypophysectomized rats.40 In the present study, we have demonstrated that the overexpression of GH-R mRNA occurred in parallel with or shortly after the peak of IGF-I mRNA. The reason for the elevated GH-R expression is not clear, although it may indicate that the IGF-I gene expression response, as well as the subsequent protein synthesis, is not enough to create a sufficient adaptive hypertrophy. Supportive evidence for this notion was presented in a recent study by Isgaard and coworkers,15 who found increased levels of both IGF-I and GH-R mRNA in the heart, with a temporal relationship between these factors in response to volume overload.
Circulating neurohormonal factors such as Ang II and norepinephrine are elevated early in both models used in the present report. These factors are important contributors in creating an optimal milieu to facilitate the adaptational growth response in the tissue. However, circulating levels of Ang II are not likely to solely regulate the IGF-I tissue levels, because both types of vessels in the two experimental models are exposed to similar concentrations of circulating Ang II, which would result in a general overexpression of IGF-I mRNA. Several studies have documented increased local activity of the components of the renin-angiotensin system, such as an increased aortic angiotensin-converting enzyme mRNA by Northern blot analyses in renal hypertension41 as well as increased angiotensin-converting enzyme activity in the pressure-overloaded left ventricle.42 Thus, increased autocrine/paracrine activation of Ang II may be involved in causing vascular hypertrophy directly, either dependently or independently of systemic factors and hemodynamic stimuli.
In the present study, we have characterized the extent and time course of the GH/IGF-I axis response in the caval vein and aorta in two different hemodynamic models—one with a rapid and the other with a slower onset of pressure and volume loads. Taken together, our findings indicate that increased systolic wall stress may be one factor, possibly acting in concert with neurohormonal activation, that augments gene expression of both GH-R and IGF-I mRNAs and IGF-I protein contents in the vessel wall. These factors are associated with the alteration in vascular structure in the vessel wall. The early and more pronounced increase of the GH/IGF-I axis in the caval vein of the ACF groups may suggest that the magnitude of wall stress correlates with the growth response.
Selected Abbreviations and Acronyms
|2K1C||=||two-kidney, one clip|
|Ang II||=||angiotensin II|
|GH-R||=||growth hormone receptor|
|IGF-I||=||insulin-like growth factor-I|
The present study was supported by the Swedish Medical Research Council (9047, 11133, 2265, 2855); Kabi Pharmacia, Stockholm; Göteborg Medical Society; the Swedish Medical Society; the Swedish Society for Medical Research; the Lundberg Foundation; the Magnus Bergwall Foundation; the Wiberg Foundation; the Novo Nordisk Foundation; the Anna Ahrenberg Foundation; the Heart and Stroke Foundation of Ontario; and PMAC/HRF-MRC of Canada. The authors wish to thank Vuk Kujacic and Entela Bollano for expertise with the ultrasound technique, Britt Gabrielsson for expertise with the RNase protection, and Jim Banting and Corry Smallgange for help with the telemetry technique. The authors also wish to thank Dr Louis F. Underwood for the gift of the IGF-Ib antiserum.
Reprint requests to Peter Friberg, MD, PhD, Department of Physiology, Institute of Physiology and Pharmacology, University of Göteborg, Medicinaregatan 11, S-413 90 Göteborg, Sweden. E-mail email@example.com.
- Received May 17, 1996.
- Revision received June 27, 1996.
- Revision received August 5, 1996.
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