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Clinical Studies |
Departments of Internal Medicine and Physiology, University of Manitoba, Winnipeg, Manitoba, Canada R3A 1R9
Address all correspondence and requests for reprints to: Dr. B. L. Gregoire Nyomba, Section of Endocrinology and Metabolism, Health Sciences Center, 820 Sherbrook Street GG449, Winnipeg, Manitoba, Canada R3A 1R9.
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Abstract |
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 Top Abstract IntroductionSubjects and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Recently, IGFBP-3 has been shown to undergo partial proteolysis under certain conditions, and this is believed to increase circulating free IGFs and promote IGF bioavailability (5). However, IGFBP-1 is the sole binding protein that has been unequivocally demonstrated to modulate the hypoglycemic activity of the IGFs (6, 7, 8). Serum IGFBP-1 levels are reduced in the immediate postprandial period and display an inverse relationship with serum insulin levels. Injection of human IGFBP-1 to rodents increases blood glucose levels (7), whereas transgenic mice expressing IGFBP-1 develop hyperglycemia (8). These studies suggest that the IGFs represent an important tonic insulin-like activity in vivo and that hyperglycemia may develop due to excessive IGFBP-1 buffering of free IGFs.
Under physiological circumstances, IGFBP-1 is thought to regulate IGF levels in response to acute metabolic changes, e.g. in response to food and exercise, in addition to shuttling these peptides from blood to target tissues (4). IGF-I appears to be more sensitive to metabolic status than IGF-II, but the total IGF-I concentration does not display short term variations related to glucose metabolism (4, 9, 10), and it is not known whether the free IGF-I level varies during short term hyperglycemia or hyperinsulinemia. The present study was undertaken to examine the relationship of serum free IGF-I with glucose homeostasis and to evaluate the effects of acute changes in plasma glucose and insulin levels on free IGF-I, IGFBP-1, and IGFBP-3.
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Subjects and Methods |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Eleven nondiabetic subjects [five men and six women; aged 29 \ 3 yr; body mass index, 24 \ 1 kg/m2 (mean \ SE)] were studied. Based on history and physical examination, these subjects were healthy and were taking no medication. Equal numbers of women were in the first and the second phase of their menstrual cycles. The study was approved by the ethics committee of the Faculty of Medicine, University of Manitoba. Informed written consent was obtained from all subjects before their participation.
Experimental protocol
An insulin-modified, frequently sampled iv glucose tolerance test (FSIGT) was performed after an overnight fast (11). A bolus of 50% glucose (0.3 g/kg) was injected at time zero, and an iv bolus (0.05 U/kg) of regular insulin (Humulin R, Eli Lilly Co., Indianapolis, IN) was given at 20 min. Blood samples were drawn at -30, -15, 0, 1, 2, 3, 4, 5, 6, 7, 8, 10, 12, 14, 16, 19, 22, 23, 24, 25, 27, 30, 40, 50, 60, 70, 80, 90, 100, 120, 150, and 180 min for the determination of glucose, insulin, total IGF-I, free IGF-I, IGFBP-1, and IGFBP-3. The samples were separated by centrifugation immediately after clotting, and serum was stored in aliquots at -20 C until assayed. Assays were performed in duplicate less than 4 weeks after sample collection, and in any assay, all blood samples from two to four subjects were analyzed in the same assay.
Serum analysis
Free IGF-I, total IGF-I, IGFBP-1, and IGFBP-3 were determined with two-site coated tube IRMAs, using commercial kits (Active, Diagnostic Systems Laboratories, Webster, TX), according to the manufacturer’s instructions. Total IGF-I was measured without prior extraction because this assay is less tedious and has been demonstrated to yield results similar to those of extractional assays (12). The respective minimal detection limits for free IGF-I, total IGF-I, IGFBP-1, and IGFBP-3 were 0.03, 3, 0.2, and 0.5 µg/L, respectively. Intraassay coefficients of variation were 6%, 5%, 5%, and 3%, whereas interassay coefficients of variation were 9%, 6%, 7%, and 4%, respectively. According to the manufacturer’s specifications, the IGFBP-1 assay primarily measures phosphorylated IGFBP-1 and may theoretically underestimate the total serum concentration of this protein. To determine the impact, if any, of this differential recognition of the various IGFBP-1 phosphoforms in our experiments, we also used a phosphorylation-indifferent total IGFBP-1 assay from the same manufacturer on 62 samples collected from 2 individuals during FSIGT. The fluctuations in the IGFBP-1 concentrations during the FSIGT did not differ between the 2 assays (data not shown). The ratio of IGFBP-1 to total IGFBP-1 was 0.34 \ 0.01 and remained constant after both glucose and insulin administration, in agreement with previous studies that showed that insulin has no effect on IGFBP-1 phosphorylation status (13).
Serum insulin was determined by a double antibody RIA (Pharmacia Canada, Baie D’Urfe, Quebec). Serum glucose was measured using a YSI 2300 STAT PLUS glucose analyzer (Yellow Springs, OH).
Data analysis
The insulin sensitivity index (SI) and glucose effectiveness (SG) were calculated using the MINMOD program (copyright R. N. Bergman). The acute insulin response to glucose (AIRG) was calculated as the area under the insulin curve from 0–10 min using the trapezoid rule. Friedman’s two-way ANOVA was applied to the overall comparison of the repeated measurements, followed by pairwise comparison with Wilcoxon’s test for related samples. Spearman’s rank test was used to derive univariate correlation coefficients. Partial correlations were computed on ranks (14). The results were considered significant at P < 0.05. Data are shown as the mean \ SE.
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Results |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Table 1
shows the characteristics of the subjects.
Women had slightly higher IGFBP-3 concentrations (P <
0.05) and tended to have higher fasting insulin and IGF-I levels
(P < 0.10) than men. Men and women were otherwise
comparable, and there were no differences related to menstrual phase
(not shown).
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Glucose injection at time zero resulted in a rapid rise in serum glucose from a basal value of 4.4 \ 0.1 mmol/L to a peak of 13.1 \ 1.1 mmol/L at 1 min postinjection. Serum glucose then gradually declined. Insulin injection at 20 min led to an acceleration of the rate of glucose fall, which was clearly evident by 40 min, resulting in an undershoot (nadir, 2.4 \ 0.2 mmol/L at 60 min), and serum glucose returned to fasting levels by 100 min. Serum insulin rose from 37.7 \ 3.2 pmol/L to a peak of 230.3 \ 46.9 pmol/L by 2 min after glucose injection and declined gradually thereafter. Injection of insulin at 20 min resulted in an elevation in serum insulin to 1,660.7 \ 296.8 pmol/L at 22 min, returning to basal levels by 60 min. The AIRG was 1532.6 \ 304.6 pmol/L·min. The SI and SG were 1.83 \ 0.39 x 10-4 min-1/pmol and 2.88 \ 0.31 min-1, respectively.
The time courses of free IGF-I, total IGF-I, IGFBP-1, and IGFBP-3
during the FSIGT are shown in Fig. 1. Total IGF-I levels
remained constant throughout the experiment. Free IGF-I declined from
0.89 \ 0.21 µg/L (basal) to 0.71 \ 0.17 µg/L at 19 min
and 0.59 \ 0.15 µg/L at 40 min. Thereafter, free IGF-I levels
did not change significantly until the end of the experiment at 180
min. After glucose injection, serum IGFBP-1 remained at basal levels
for about 50 min. IGFBP-1 fell steadily thereafter, resulting in an
undershoot by 100 min, then sharply increased by about 3-fold by the
end of the experiment at 180 min. IGFBP-3 levels remained constant for
about 30 min, then they increased linearly thereafter to about 20%
above basal values.
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In the fasting state, free IGF-I was positively correlated with
SI (r = 0.52; P < 0.005) and
SG (r = 0.37; P < 0.05) and inversely
correlated with glucose (r = -0.51; P < 0.005)
and IGFBP-1 (r = -0.65; P < 0.001), but not with
AIRG or IGFBP-3. A significant inverse correlation of
free IGF-I with glucose was also found after glucose load (r =
-0.33; P < 0.001), but not after insulin injection
(r = 0.02; P = NS). There was a hyperbolic
relationship between free IGF-I and IGFBP-1, which was significant
at each time point during the FSIGT, except from 120–180 min,
i.e. during the IGFBP-1 overshoot (Fig. 2).
Total IGF-I was also inversely correlated with glucose (r =
-0.37; P < 0.05) and IGFBP-1 (r = -0.42;
P < 0.025) and positively correlated with IGFBP-3
(r = 0.59; P < 0.001), but not with
SI, SG, or AIRG. The relationships
of free IGF-I remained significant after controlling for total IGF-I.
Except for IGFBP-3, however, the relationships of total IGF-I were
dependent on free IGF-I. There was no correlation between IGFBP-1 or
IGFBP-3 and serum glucose.
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Discussion |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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IGFBP-3 levels increased linearly after insulin administration in a pattern that mirrored the decline of free IGF-I. There were, however, no measurable changes in total IGF-I levels. The source of IGFBP-3 and the mechanisms underlying its stimulation by insulin are unclear. The synthesis of this protein takes place in various tissues and is under GH control directly or indirectly via IGF-I (18, 19). However, Scharf et al. (20) recently observed a stimulation of IGFBP-3 synthesis by insulin in mixed liver cell cultures. In addition, insulin stimulated the synthesis of the acid-labile subunit, an essential component of the circulating 150-kDa IGFBP-3-IGF complex (4). Conceptually, insulin might also augment the IGFBP-3 level by inhibiting the IGFBP-3 protease, whose activity has been reported to increase in untreated diabetic patients and to decrease after several days of insulin treatment (5). Regardless of the mechanism, an increase in IGFBP-3, as found in the current study, would explain at least in part the decline in free IGF-I induced by insulin.
Another possibility is that insulin suppresses free IGF-I through its effect on IGFBP-1. A bolus insulin injection decreased IGFBP-1 levels with a nadir at 100 min, which is in close agreement with studies of insulin’s effect in cell cultures (21) or human studies using continuous insulin infusion. Brismar et al. (22) demonstrated that insulinopenia in diabetic patients was associated with high IGFBP-1 and low total IGF-I levels, and that insulin infusion in these patients inhibited IGFBP-1 production by the liver while increasing IGF-I production. These researchers hypothesized that this is a mechanism by which insulin increases IGF-I bioavailability. This hypothesis is supported by our finding in the current study and that of Graubert et al. (23) in diabetic rats of a hyperbolic relationship between free IGF-I and IGFBP-1 levels. In our study, however, this relationship no longer existed during the IGFBP-1 overshoot at the end of the FSIGT, which is the time when the lowest free IGF-I levels were found. This IGFBP-1 overshoot was detected using both phosphorylation-sensitive and phosphorylation-insensitive assays and, therefore, cannot be an artifact of the assay caused by a change in IGFBP-1 phosphorylation status. The rise in IGFBP-1 in the current study was comparable to what Westwood et al. (13) observed using a 4-fold higher insulin dose in healthy adult men. In their study, however, IGFBP-1 took 5 h to rise after insulin injection. The observed sudden elevation in IGFBP-1 may be the explanation for the alteration in the inverse relationship between free IGF-I and IGFBP-1 at the end of the FSIGT. Because the time courses of free IGF-I and IGFBP-1 were so different, insulin-induced changes in the IGFBP-1 levels per se cannot account for the decrease in free IGF-I levels following insulin injection. It is also unlikely that the decrease in free IGF-I in our study is caused by fasting. These conclusions are in agreement with recent findings by Bereket et al. (24) that an overnight fast is associated with a marked elevation in IGFBP-1 without alteration in the circulating levels of free IGF-I. However, because insulin has been shown to stimulate IGFBP-1 transport across the endothelial wall (25), it is possible that this clearance of IGFBP-1 or of the IGF-I-IGFBP-1 complex results in a new equilibrium, with lower free IGF-I levels.
The suppression of free IGF-I by insulin found in this study was unexpected in view of previous reports that insulin increases IGF-I production in diabetic patients and animals and in tissue cultures (26, 27, 28). However, there are suggestions that insulin might also decrease IGF-I levels. Giacca et al. (29) detected a slight decrease in total IGF-I levels at the end of a 3-h insulin infusion in depancreatized dogs, and Lang et al. (30) found an increase in IGF-I uptake by the legs after 7 days of insulin infusion in burn patients. As these were catabolic, presumably insulin-resistant, subjects, it may be anticipated that tissue uptake of IGF-I in normal subjects would be higher or faster. In the presence of normal insulin sensitivity, therefore, it is conceivable that a disproportionately increased uptake of IGF-I relative to its production would result in decreased serum levels of free IGF-I, as discovered in this study.
The prospect of an insulin-stimulated tissue uptake of IGF-I is interesting because this would suggest a synergism among these hypoglycemic hormones. We found in normal subjects a negative correlation between free IGF-I and glucose levels, which was most significant in the fasting state and tended to disappear with increasing insulin levels, i.e. after glucose and insulin administration. In addition, fasting free IGF-I was positively correlated with SI. These results point to a role for free IGF-I to regulate glucose metabolism, especially in the fasting state, and to a link between circulating free IGF-I and insulin action. These observations were not unexpected, as treatment of type II diabetic patients with IGF-I has been shown to increase insulin sensitivity and improve metabolic control (31).
In summary, circulating free IGF-I concentrations in normal subjects change inversely with fasting blood glucose and IGFBP-1 levels. Insulin stimulates IGFBP-3 accumulation and differentially suppresses free IGF-I and IGFBP-1 levels, altering their interrelation as well as their relationship with serum glucose. Both insulin and IGF-I seem to concur to maintain normal glucose levels, and IGF-I might exert a tonic effect on glucose homeostasis, especially in the absence of insulin, e.g. in the fasting state.
Received January 23, 1997.
Revised March 20, 1997.
Accepted March 31, 1997.
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) at time zero and
insulin (•) at 20 min. Values are shown as the mean ±
SE percentage of the basal value.