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Original Studies |
Institute of Experimental Clinical Research, Medical Research Laboratories, Aarhus University Hospital, Denmark
Address correspondence and requests for reprints to: Jan Frystyk, MD, PhD, Institute of Experimental Clinical Research, Aarhus Kommune Hospital, Nørrebrogade 44, DK-8000 Aarhus C, Denmark.
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Abstract |
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Glucose, insulin, and GH followed the expected patterns and had regained baseline levels at 270 min. Total IGF-I and free and total IGF-II remained unaltered. IGFBP-1 decreased significantly by 37–52% (P < 0.05) from 150 to 210 min, whereafter the concentration gradually increased by 75% to a level that tended to be above baseline (P = 0.052). Free IGF-I decreased by 29–38% (P < 0.05) at the end of the study (270–330 min). IGFBP-1 was inversely correlated to free IGF-I at baseline (r = -0.57; P < 0.05), as well as during the OGTT (r = 0.66; P < 0.0001). In contrast, free IGF-II was not correlated to IGFBP-1. Insulin, but not free IGF-I, correlated significantly with serum glucose (P < 0.05).
These results extend our previous findings of an inverse correlation between free IGF-I and IGFBP-1 in cross-sectional studies to include longitudinal observations, and thus further substantiates the hypothesis that IGFBP-1 is an important determinant of free IGF-I in vivo. Significant changes in free IGF-I were observed only in the late postprandial phase, when glucose and insulin were fully normalized, demonstrating that free IGFs probably do not participate in glucoregulation to any significant degree during an oral glucose load in healthy subjects.
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Introduction |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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IGFBP-1 binds only a minor portion of the circulating IGFs, but has received considerable attention because of its unique properties. IGFBP-1 is the only one of the six IGFBPs showing a rapid dynamic regulation in human plasma (4). The production of IGFBP-1 by the liver is inversely regulated by portal levels of insulin (5), and circulating levels of IGFBP-1 exhibit significant changes during the day (2, 6, 7). Thus, IGFBP-1 is believed to regulate IGF-bioactivity in vivo in relation to fuel supply (2, 4). In support of this, we found inverse relations between circulating levels of free IGF-I and IGFBP-1 in overnight fasted healthy adults (8), after extended fasting (3), and in nonfasted healthy children (9). We have, however, primarily investigated cross-sectionally obtained samples, and studies of the individual time course of changes in IGFBP-1 and free IGF-I are needed to evaluate the physiological association between the peptides.
The tight connection between IGFBP-1 and fuel supply and the hypoglycemic insulin-like activity of the IGFs suggest that the IGF-system (free IGF and IGFBP-1 in particular) may be involved in glucose homeostasis (10). This hypothesis has been supported by recent experimental in vivo studies: in rats receiving bolus injections of IGFBP-1 (11) and in transgenic mice over-expressing IGFBP-1 (12), the presence of high circulating levels of IGFBP-1 resulted in fasting hyperglycemia. In humans, however, the possible participation of the IGF-system in glucose homeostasis is not established.
The aim of the present study was to examine 1) the time course of changes in serum levels of free IGFs and IGFBP-1 during an oral glucose tolerance test (OGTT), which is known to change circulating levels of IGFBP-1 (7, 13); and 2) the possible participation of the IGF-system in glucose homeostasis.
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Subjects and Methods |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Thirteen healthy nonobese subjects (9 males and 4 females, age 23–36 yr (range), weight 56–80 kg, height 1.74-I.92 m, body mass index (BMI) 18.5–25.4 kg/m2) were admitted to the hospital on the morning of the study following an overnight fast, starting at 2200 hours. An iv cannula was placed in the antecubital fossa. After a 30 min rest the first blood sample (0 min, 10 mL) was collected, followed by ingestion within 5 min of 75 g glucose dissolved in 150 mL water. Blood samples were collected every 30 min for the next 330 min, and the subjects remained recumbent throughout the study. The study was approved by the local ethics committee in Aarhus Council, Denmark, and all subjects gave informed consent to participate.
IGF-I and IGF-II were determined by use of two in-house monoclonal, noncompetitive assays, as previously described (14). Serum total IGF-I and IGF-II were determined in duplicates in acid ethanol serum extracts (final dilution: 1 in 1066). The lower detection limit in serum is then 3 µg/L (IGF-I) and 11 µg/L (IGF-II), and the cross reactivity in heterologous assays is <0.0002%. Intraassay and interassay coefficients of variation (CV) averaged 5% and 10%, respectively. Serum free IGF-I and IGF-II were determined by ultrafiltration and centrifugation as previously described (3). Amicon YMT 30 membranes and MPS-1 supporting devices were used (Amicon Division, W.R. Grace and Co., Beverly, MA). Before centrifugation, serum samples were diluted (1 in 11) in Krebs Ringer buffer, which had been adjusted to pH 7.4 by airing with CO2. From each dilution an aliquot of 600 µL was applied to the membranes, incubated (30 min at 37 C), and centrifuged (1500 RPM at 37 C) (Hettich Zentrifugen, Tuttlingen, Germany). Ultrafiltrates were collected in 5 mL polyethylene tubes that, before centrifugation, were coated with human serum albumin. In the ultrafiltrates the lower detection limit was 30 ng/L (IGF-I) and 110 ng/L (IGF-II). To compare concomitant changes of free IGF-I and free IGF-II each free fraction was determined in triplicate in separate ultrafiltrates. Care was taken to analyze each subject within the same assay, and following ultrafiltration plus analysis the intraassay coefficient of variation (CV) averaged 16% (free IGF-I) and 17% (free IGF-II).
Serum GH, IGFBP-3, and IGFBP-1 were measured by commercial assays: GH by a noncompetitive assay (DELFIA GH, Wallac Oy, Turku, Finland) (15), IGFBP-3 by radioimmunoassay (Diagnostic System Laboratories Inc., Webster, TX), and IGFBP-1 by enzyme-linked immunosorbent assay (ELISA) (Medix Biochemica, Kainiainen, Finland). This IGFBP-1 kit measures the non- and lesser phosphorylated isoforms of IGFBP-1 (13, 16).
Serum insulin was determined by a polyclonal radioimmunoassay using recombinant human (rh) insulin and I125-rh-insulin as calibrator and tracer (Novo Nordisk A/S, Bagsværd, Denmark), and serum glucose by the glucose-oxidase method.
Statistics
We analyzed changes from baseline by use of repeated measures statistics. Parametric data (total IGF-I, total IGF-II, and free IGF-I) were analyzed using repeated analysis of variance (ANOVA) followed by Student’s t test using Bonferroni’s confidence limits. Nonparametric data (free IGF-II, IGFBP-1, GH, glucose, and insulin) were analyzed using Friedman’s paired multiple rank sum test followed by Dunn’s test for multiple comparisons. Correlations between data were analyzed by linear regression analysis. A P-value < 0.05 was considered statistically significant. All values are mean ± SEM.
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Results |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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At the start of the study all subjects had normal fasting levels of serum glucose (5.0 ± 0.1 mmol/L), GH (1.0 ± 0.6 µg/L), insulin (8.7 ± 0.7 mU/L), IGFBP-1 (2.7 ± 0.7 µg/L), total IGF-I (235 ± 15 µg/L), free IGF-I (980 ± 140 ng/L), total IGF-II (875 ± 28 µg/L), and free IGF-II (1020 ± 100 ng/L). At baseline, free IGF-I, but not free IGF-II, was inversely correlated with IGFBP-1 (after log transformation; r = -0.57; P < 0.05).
Changes during the OGTT.
Serum glucose had increased 30 min after oral glucose
(P < 0.05); Fig. 1
. This
was followed by a gradual decrease, and serum glucose was significantly
(P < 0.05) below baseline levels from 150 to 180 min,
after which baseline levels were reattained. Serum insulin and GH were
significantly (P < 0.05) elevated from 30 to 90 min
and from 210 to 240 min, respectively, when compared with baseline
levels (Fig. 1). Serum total IGF-I and IGF-II did not change during the
OGTT, being 241 ± 16 µg/L and 891 ± 35 µg/L,
respectively.
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, upper panel).
Serum free IGF-I decreased significantly by 29–38% (P
< 0.05) towards the end of the study, from 980 ± 140 ng/L at
baseline to 700 ± 100 ng/L (270 min), 610 ± 80 ng/L (300
min), 660 ± 80 ng/L (330 min; Fig. 2, upper panel).
Thus, the changes in IGFBP-1 were not accompanied by opposite changes
in free IGF-I until the late postprandial phase. Nevertheless, in 10
out of 13 subjects serum free IGF-I was significantly inversely
correlated with serum IGFBP-1 (-0.87 < r < -0.59;
0.0005 < P < 0.05). The 13 individual regression
lines of serum free IGF-I as a function of serum IGFBP-1 are depicted
in Fig. 2 (lower panel). We have also shown the common
regression line, which was calculated as the mean of the 13 individual
regression lines; Fig. 2 (lower panel). The slope of this
line was significantly different from zero (slope = -122 ±
37; P = 0.007) as was the r-value (r = -0.66
± 0.05; P < 0.0001).
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To study the possible role of free IGF-I in glucose regulation during the OGTT, serum levels of free IGF-I and insulin were compared with those of serum glucose by linear regression analysis. First however, data was log-transformed to ensure that the residuals fulfilled the criteria for normality. A significant positive correlation between serum levels of insulin and glucose was observed in 9 out of 13 subjects (0.67 < r < 0.96; 0.0001 < P < 0.02). The slope of the common regression line was significantly different from zero (slope = 0.182 ± 0.021; P < 0.0001), as was the r-value (r = 0.78 ± 0.04; P < 0.0001). In contrast, serum free IGF-I was only significantly correlated with serum glucose in 3 subjects (0.58 < r < 0.62; 0.03 < P < 0.05), and both the slope and r-value of the common regression line were indistinguishable from zero (data not shown).
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Discussion |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Serum IGFBP-1 was significantly suppressed 150 to 210 min after oral glucose, whereafter the concentration returned towards levels that were not statistically different from those at baseline (P = 0.052). However, only the latter caused significant changes in serum free IGF-I, which decreased by 0.32 µg/L. We believe that this is the result of the magnitude of changes in IGFBP-1. Whereas the initial reduction in IGFBP-1 averaged 1.2 µg/L, at the time when the changes in free IGF-I became significant, IGFBP-1 had increased by 2.2 to 3.0 µg/L when compared with levels at 210 min. Considering that the initial changes in IGFBP-1 were considerably less pronounced than those observed at the end of the study, we believe that the reduction in serum IGFBP-1 following oral glucose was too small to cause a detectable increase in serum free IGF-I.
Our results resemble those recently published by Bereket, et
al. (17). They compared overnight fasting levels of free IGF-I and
IGFBP-1 with those 4 h after a carbohydrate-rich breakfast in 17
healthy subjects. The 4-h level of IGFBP-1 was approximately 5-fold
lower than the fasting level. Free IGF-I, determined by use of
ultrafiltration according to our previous paper (3), did not change,
but averaged 1.4 µg/L in both situations. On this basis the authors
suggested that changes in IGFBP-1 concentrations within the
physiological range were not sufficient to affect circulating free
IGF-I, and that IGFBP-1 did not change IGF-bioactivity by limiting free
IGF-I availability (17). The results of the present study resemble
those of Bereket et al. (17), i.e. levels of free
IGF-I were unchanged up to 4 h after ingestion of glucose, despite
significant changes in IGFBP-1 (please see Fig. 2
, upper
panel). The decrease we observed in free IGF-I occurred 4 h
after oral glucose, at a time when IGFBP-1 began to increase to levels
that tended to be above baseline (P = 0.052). However,
the longitudinal design of our study with 12 consecutive samples in 13
subjects clearly showed that free IGF-I was inversely correlated with
IGFBP-1. Furthermore, the findings of the present study are in
accordance with our previous observations of an inverse correlation
between free IGF-I and IGFBP-1 in cross-sectionally collected
samples from healthy adults (8) and children (9), patients suffering
from ischemic heart disease (18), and GH deficient subjects (19).
Serum from healthy, nonpregnant subjects contains two isoforms of
IGFBP-1: a major fraction (
90%) of highly phosphorylated IGFBP-1
(p-IGFBP-1), and a minor fraction (10%) of nonphosphorylated
IGFBP-1 (np-IGFBP-1) (16). In the present study we have used a
commercially available kit that measures np-IGFBP-1 only (13, 16), and
although one may argue that this isoform is less important than
p-IGFBP-1, the observed inverse correlation between free IGF-I and
np-IGFBP-1 clearly suggests a role for np-IGFBP-1 in the regulation of
free IGF-I in vivo. However, further studies are needed to
compare changes in p-IGFBP-1 and np-IGFBP-1 with those of free
IGF-I.
Of notice is that free IGF-II was not correlated to IGFBP-1, in accordance with our previous findings (8, 20, 21). This seems to conflict with recent observations that np-IGFBP-1 has similar affinity for IGF-I and IGF-II in vitro (22). One explanation could be that the higher affinity of IGFBP-2, -3, -5, and -6 for IGF-II (23, 24) will counteract the effects of IGFBP-1 on free IGF-II, and favor dynamic interactions between free IGF-I and IGFBP-1.
The present paper also aimed to evaluate the possible participation of
the IGF-system in glucose homeostasis. The rapidly changing serum
levels of IGFBP-1 are tightly connected to the portal levels of insulin
(5), and IGFBP-1 has been suggested to modulate the short-term
insulin-like activity of the IGF-system (10). In humans receiving
short-term treatment with rh-IGF-I the insulin-like hypoglycemic
activity of IGF-I has been estimated to be 5–6% of that of insulin
(25, 26). In accordance, we found that at euglycemia the insulin-like
activity of free IGF-I in healthy subjects treated for 3 days with
rh-IGF-I would correspond to 4% of that of insulin (27). Using this
result it can be estimated that the insulin-like activity of free IGF-I
corresponded to about 1.0 mU/L at baseline and decreased to 0.65 mU/L
towards the end of the study. The contribution of IGF-II on the
hypoglycemic insulin-like activity has not been studied in man, but the
insulin receptor has a higher affinity for IGF-II than for IGF-I (28),
indicating that the insulin-like effect of free IGF-II equals at least
1.0 mU/L of insulin. Thus, in the present study the estimated glucose
lowering effect of circulating free IGF-I plus free IGF-II corresponded
to 1.5–2 mU/L of insulin. However, the changes in serum free IGF-I
took place when serum glucose and insulin (8 mU/L at baseline and at
the end of the study) were fully normalized. Furthermore, we observed
no significant correlation between serum free IGF-I and serum glucose,
whereas the latter was positively correlated to serum insulin. This
indicates that, during the first 4 h after oral glucose, the
contribution of the IGF-system on glucose regulation is of minor
physiological importance. On the other hand, our results do not leave
out the possibility that, in fasting or late postprandial serum samples
from healthy subjects, free IGF-I plus IGF-II may exert a tonic glucose
lowering effect, accounting for as much as 20% of the total
insulin-like activity. Similar findings have been obtained in rats (11)
and transgenic mice (12).
In conclusion, during an oral glucose tolerance test in healthy subjects we observed a significant inverse correlation between serum free IGF-I and IGFBP-1. This supports the view that IGFBP-1 is an important determinant of free IGF-I and presumably of IGF-I bioactivity in vivo. However, the glucose challenge did not significantly increase the circulating levels of free IGF-I in the early postprandial phase, despite a significant decrease in serum IGFBP-1. This indicate that the IGF-system does not participate in the acute glucoregulation following ingestion of 75 g of glucose. Interestingly, serum free IGF-II seems to be less influenced by IGFBP-1, suggesting that the peptides may have different roles in short-term fuel balance, as studies of serum total IGF-I and IGF-II have also proposed (29).
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Acknowledgments |
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Footnotes |
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Received February 26, 1997.
Revised June 5, 1997.
Accepted June 16, 1997.
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References |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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; right Y-axis) and serum GH (
; left
Y-axis) during the OGTT. Data are mean ± SEM. *:
P < 0.05, when compared with baseline (t = 0
min).