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Original Studies |
Institut National de la Santé et de la Recherche Médicale (C.L., M.B.), Unité 515, Département d’Endocrinologie et Diabétologie (F.D.), Hôpital Saint Antoine, Assistance Publique-Hôpitaux de Paris, Université Paris VI, 75571 Paris Cedex 12, France
Address all correspondence and requests for reprints to: M. Binoux, Institut National de la Santé et de la Recherche Médicale, Unité 515, Hôpital Saint Antoine, 184, rue du Faubourg Saint Antoine, 75571 Paris Cedex 12, France. E-mail: u515{at}st-antoine.inserm.fr
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Abstract |
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Because GH and insulin control the hepatic production and plasma concentrations of IGF-I and IGFBP-3, we set out to determine whether variations in the secretion of the two hormones are involved in the regulation of IGFBP-3 proteolysis. The study included adult populations of 36 healthy subjects, 23 hypopituitary patients untreated with GH, 43 acromegalics (13 untreated), 42 insulin-treated type 1 diabetics [insulin-dependent diabetes mellitus (IDDM)] patients, and 50 type 2 diabetics [non-IDDM (NIDDM)] patients, 22 of whom were insulin-treated and the remaining 28 treated with sulfonylurea and/or metformin).
Unlike IGF-I and (to a lesser extent) total IGFBP-3 levels, which decline with age, percent proteolyzed IGFBP-3 seemed relatively stable. In healthy adults, the mean ± SEM was 29.4 ± 1.9 for subjects less than 45 yr old and was slightly (but not significantly) lower, 25.7 ± 3, for those of more than 45 yr. There was no difference between male and female subjects.
In GH-deficient patients, despite severely depressed IGF-I levels, percent proteolyzed IGFBP-3 and IGF-I/intact IGFBP-3 ratios were within the normal range. Among acromegalics, percent proteolyzed IGFBP-3 was elevated: 36.6 ± 3.3 for patients of less than 45 yr, 33.3 ± 3.2 for patients of more than 45 yr (P = 0.02 vs. healthy subjects). Consequently, the effects of excessive IGF-I synthesis are exacerbated by the enlarged exchangeable fraction of IGFBP-3-bound IGF-I. There was no significant difference in percent proteolyzed IGFBP-3 between GH-deficient patients before and after GH treatment or between treated and untreated acromegalics.
In IDDM patients, the means for percent proteolyzed IGFBP-3 were higher than those in healthy adults: 36.7 ± 3.7 (P = 0.03) and 31.3 ± 3.3 for subjects of less than 45 and more than 45 yr, respectively. In NIDDM patients, all of whom were more than 45 yr old, the means were 35.2 ± 2.5 (P = 0.02) for insulin-treated patients and 33 ± 2.5 for the group treated orally. Among the diabetics, increased IGFBP-3 proteolysis resulted in an IGF-I/intact IGFBP-3 ratio that was normal for IDDM patients of less than 45 yr and above normal (P = 0.01) for the others.
Percentage proteolyzed IGFBP-3 and the IGF-I/intact IGFBP-3 ratio were inversely related to body mass index in IDDM patients (r = -0.42, P = 0.008; and r = -0.31, P = 0.05, respectively) and to percentage glycosylated hemoglobin in all insulin-treated diabetics (r = -0.25, P = 0.05; and r = -0.33, P = 0.008, respectively). There was also an inverse relationship between IGF-I/intact IGFBP-3 ratios and IGFBP-1 levels in healthy adults (r = -0.39, P = 0.03) and orally treated NIDDM patients (r = -0.37, P = 0.05).
Percentage proteolyzed IGFBP-3 was positively correlated to total IGFBP-3 in healthy adults (r = 0.65, P = 0.0001) and in all the groups of patients. It was negatively correlated to IGF-I/total IGFBP-3 in healthy subjects (r = -0.40, P = 0.02) and diabetics (r = -0.30, P = 0.005). This suggests an autoregulatory mechanism controlling the bioavailability of IGFBP-3-bound IGF-I in the 140-kDa complexes. In the pathological conditions studied here, regulation of IGF-I bioavailability by limited proteolysis of IGFBP-3 contributes toward an appropriate adaptation to insulin deficiency and/or resistance but not to disturbances of GH secretion.
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Introduction |
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The hepatic origin of IGF-I in the bloodstream was definitively demonstrated by liver-specific gene deletion in mice, which results in marked reduction of its plasma concentrations (5). In the face of normal postnatal growth in these mice, reservations were expressed as to the role of liver-derived IGF-I (5, 6). Compensatory IGF-I secretion by other organs in response to the large increase in serum GH levels in these animals would, however, not exclude the possibility of its playing an important endocrine role in the normal state. Proof of this endocrine role is provided by the changes in the bioavailability of serum IGF-I to its target tissues. In normal circumstances, most IGF-I is sequestered by IGFBP-3 associated with the acid-labile subunit in 140-kDa complexes with a long half-life. In GH deficiency, IGF-I is shifted toward the 40-kDa IGFBP-IGF complexes that are capable of crossing the capillary endothelium (7). In addition, limited proteolysis of IGFBP-3 by serine proteases, by which IGF-I release from the 140-kDa complexes is accelerated, constitutes a further mechanism controlling IGF-I bioavailability (7, 8, 9). This mechanism is probably essential, but its regulation remains unknown. The ligand immunofunctional assay (LIFA) described in our previous paper (10) provides for measurement specifically of intact IGFBP-3, as opposed to its proteolytic fragments that are indiscriminately detected by the IGFBP-3 RIAs and immunoradiometric assays (IRMAs) in current use. In the present study, we combined the LIFA with an IRMA for total IGFBP-3, from which the proportions of proteolyzed IGFBP-3 could be quantified in plasma. In addition, with measurement of total IGF-I concentration by RIA, the ratio of IGF-I to intact IGFBP-3 could be determined as an index of the exchangeable (dissociable) fraction of IGF-I bound to IGFBP-3. These parameters were then analyzed under physiological and pathological conditions comprising abnormalities of GH secretion (hypopituitarism, acromegaly) and of insulin secretion and/or sensitivity (types 1 and 2 diabetes). This selection of subjects was based on the notion that hepatic production of IGF-I and IGFBP-3, although controlled by GH, also depends on metabolic status and insulin secretion, both of which are involved in modulation of the transduction of the GH signal via its receptor (7, 11). The aim of the study was to determine whether these factors contribute toward regulating IGFBP-3 proteolysis.
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Materials and Methods |
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Plasma samples were obtained, between 0800 and 0900 h, from fasting subjects (all of whom had provided informed consent) and according to the rules of the hospital’s Ethics Committee. Blood was collected in tubes containing an EDTA solution (5 µmol/mL blood), on ice (10). These were centrifuged at 4 C, and plasma samples were stored at -20 C. Assays were performed within weeks or months of sampling (always less than a year). Checks were routinely done on the pool of normal adult plasma in each assay that, under the same conditions, the proportion of proteolyzed IGFBP-3 had remained stable.
The subjects, all adults, were as follows: 36 healthy volunteers (24
women, none using oral contraceptives; and 12 men), 20–65 yr old
(mean, 33); 23 hypopituitary patients, 16–49 yr old [12 of less than
30 yr, with idiopathic GH deficiency diagnosed during childhood and
investigated before and during GH therapy; 4 with pituitary adenoma; 2
with Sheehan’s syndrome; 1 with craniopharyngioma; 1 with rupture of
the pituitary stalk; and 3 with cause unknown]; 43 acromegalics,
17–76 yr old (mean, 46), 13 of whom were untreated [in the others,
treatment comprised somatostatin alone or associated with adenomectomy
(12 cases); IGF-I levels were taken as one of the criteria for
progression of the disease/prognosis]; 42 type 1 diabetics
[insulin-dependent diabetes mellitus (IDDM) patients], 19–76 yr old
(mean, 48), under insulin treatment [6 had a body mass index (BMI)
ratio of weight (kg) to height (m2) 
28];
and 50 type 2 diabetics [non-IDDM (NIDDM) patients, more than 45 yr
old (mean, 60); 28 were under treatment with sulfonylurea and/or
metformin, BMI 28 in 16 cases; 22 were under insulin treatment, oral
medication having proved inefficacious, BMI 28 in 13 cases].
Follow-up criteria systematically comprised measurement of glycemia and
glycosylated hemoglobin (HbA1c). None of these diabetics were in a
state of imbalance requiring hospitalization.
All blood samples were collected during sampling for the purposes of diagnosis or therapeutic follow-up. The last treatment before sampling was administered before the previous evening meal.
LIFA for IGFBP-3 (intact IGFBP-3)
The assay is described in detail in the previous paper (10). Briefly, IGFBP-IGF complexes were dissociated in 0.01 mol/L HCl, and their components were separated by ultrafiltration on Centricon C30 (Amicon, Epernon, France). After neutralization, duplicate aliquots of the retentates, each corresponding to an initial 3.75 µL of plasma, were incubated at three concentrations in 300 µL (total vol) phosphate buffer in Maxisorp tubes (Nunc, Roskilde, Denmark) coated with monoclonal anti-hIGFBP-3 antibody (mAb). This mAb is specific to the first 160 amino acids of IGFBP-3. After washing, to remove other IGFBPs not captured by the mAb, the tubes were incubated with 125I-IGF-I, which binds specifically to intact IGFBP-3 (and not to its proteolytic fragments).
The intact IGFBP-3 concentration is read from the standard curve established using a pool of normal adult plasmas treated identically to the unknown samples and estimated to contain 2 µg/mL intact IGFBP-3, based on a preparation of recombinant human glycosylated IGFBP-3. Sensitivity was 0.4 µL of the normal plasma pool. Intraassay variation was 3.6%; and interassay variation, below 10%.
IGF-I RIA (total IGF-I)
The assay was performed as previously reported (12). Briefly, the ultrafiltrates containing IGFs were lyophilized, then taken up in PO4BSA and incubated for 3 days in a final vol of 400 µL with the polyclonal anti-IGF-I antibody (1:120,000 dilution) and 125I-IGF-I (3,000 cpm/tube). Unknown samples were tested at three concentrations, plus one blank (without antibody), each in duplicate, so as to confirm parallelism with the standard curve. After incubation, free and bound IGFs were separated using albumin-coated charcoal. The threshold sensitivity of the assay was 1–2 ng/mL plasma. Intraassay variation was less than 5%; and interassay variation, 10%.
Immunoradiometric assay for IGFBP-3 (total IGFBP-3)
This was performed using an IGFBP-3 IRMA kit (Immunotech, Marseille, France). The mAb pool that was used specifically detects IGFBP-3 and its N-terminal proteolytic fragments (10). Each plasma sample was tested at two concentrations (0.25 and 0.50 µL) in a total vol of 450 µL per assay tube. The sensitivity threshold was 0.05 µg/mL plasma. Intraassay variation was close to 5%; and interassay variation, 10%.
Immunoradiometric assay for IGFBP-1
This assay was performed using the DSL 7800 Active IGFBP-1 IRMA kit (Diagnostic Systems Laboratories, Inc. Webster, TX). Each plasma sample was tested at two concentrations (12.5 and 25 µL per assay tube). The sensitivity threshold was 0.75 ng/mL plasma. Intra- and interassay variations were approximately 5% and 10%, respectively.
Other assays
These were performed in the Biochemistry Laboratory of Saint Antoine Hospital. Plasma glucose was determined using the glucose oxidase method; and HbA1c, by cation-exchange high-performance liquid chromatography (13) (normal range, 4–6%).
Expression of results
Intact and total IGFBP-3 concentrations were expressed in milligrams per liter.
The proportion of proteolyzed IGFBP-3 was calculated as [total IGFBP-3 - intact IGFBP-3/total IGFBP-3] x 100.
For calculation of the IGF-I/intact IGFBP-3 or IGF-I/total IGFBP-3 ratios, molar concentrations were estimated on the basis of a mass of 7.5 kDa for IGF-I and a molecular mass of 41 kDa for IGFBP-3. The latter represents the approximate mean of the two (bi- and triglycosylated) forms of IGFBP-3 (14). In normal serum, the quantities of the two forms, as estimated by Western ligand blotting, are very similar (15).
Statistics
Conventional methods, including linear regression analysis, were used. Differences between groups were studied by ANOVA. Results are presented as means ± SEM. P < 0.05 was considered statistically significant.
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Results |
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shows the means (±
SEM) for the various parameters studied in healthy subjects
and the different groups of patients. All the patients in this study
were adults, and many were of advanced age. The drop in circulating
IGF-I levels with age is well known (16, 17); and age
differences, therefore, needed to be considered. We elected to present
the data for subjects who were either below 45 yr of age or 45 yr and
above, because there were approximately equal numbers of subjects
within these age ranges among acromegalic and IDDM patients. All the
NIDDM patients were over 45.
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Percentage of proteolyzed IGFBP-3. Analysis of the changes in
percent proteolyzed IGFBP-3 in normal subjects and the different groups
of patients revealed no correlation with age. Data for healthy adults
and diabetics are shown in Fig. 1. The
means in Table 1
are slightly lower for more-than-45-yr-old normals,
acromegalics, and IDDM patients, but the differences are not
significant. There was no difference either between male and female
subjects (not shown).
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).
In hypopituitary and acromegalic patients, where disturbances of GH
secretion have major repercussions on IGF-I and IGFBP-3 levels, there
was no correlation between these variables and age. Similarly, there
was no correlation with IGF-I/intact IGFBP-3 (not shown). Means for
IGF-I/intact IGFBP-3 and IGF-I/total IGFBP-3 ratios (not shown) were
lower in patients above 45 yr of age, but the differences were not
significant (Table 1).
Differences in IGFBP-3 proteolysis and the IGF-I/intact IGFBP-3 ratio, depending on pathology
Normal subjects were compared with hypopituitary patients untreated with GH, patients with active acromegaly (IGF-I levels above the normal mean + 2 SD), IDDM patients, NIDDM patients under insulin treatment, and NIDDM patients receiving antidiabetic oral medication.
Percentage of proteolyzed IGFBP-3. In view of the absence of
significant change in IGFBP-3 proteolysis with age, data for all groups
of subjects are shown together in Fig. 2.
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, it can be seen that the means were significantly higher
than normal in acromegalics and IDDM patients less than 45 yr old, but
not those who were more than 45, and among NIDDM patients, in those
under insulin treatment but not those receiving oral medication.
IGF-I/intact IGFBP-3 ratio. Because IGF-I drops with age in
normals and diabetics, Fig. 3 shows the
data for less-than-45-yr-old normal subjects and IDDM patients,
together with those for all hypopituitary and acromegalic patients
where IGF-I synthesis depends on pathological GH secretion, which is
not (or only marginally) influenced by age; Fig. 4 shows the data for more-than-45-yr-old
normals and diabetics. IGF-I levels are also given, whereas the means
appear in Table 1.
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indicate ratios in the two age groups of GH-deficient patients that
were higher, but not significantly so, than age-matched normal
subjects. In acromegalics, both IGF-I levels and IGF-I/intact IGFBP-3
ratios were scattered across a wide range, with means approximately 3
times higher than those of normals. This would suggest that
IGF-I/intact IGFBP-3 depended more on IGFBP-3 proteolysis in
GH-deficient patients than in acromegalics. Consistent with this
notion, IGF-I/intact IGFBP-3 was positively correlated with percent
proteolyzed IGFBP-3 in hypopituitary patients (r = 0.56,
P = 0.006) but not in acromegalic patients (not
shown). Mean IGF-I levels were slightly, but significantly, lower than normals in IDDM patients less than 45 yr old (P = 0.02) but similar in those more than 45. In both age groups, IGF-I/intact IGFBP-3 ratios were, on average, higher than in normals but significantly so only among patients more than 45 yr old (P = 0.01). Among NIDDM patients, those under insulin treatment had IGF-I levels similar to those of age-matched normals; whereas, in those under oral treatment, IGF-I levels were significantly higher (P = 0.03). On average, IGF-I/intact IGFBP-3 ratios were significantly higher in NIDDM patients than in age-matched normals (P = 0.01). In all diabetics, the IGF-I/intact IGFBP-3 ratio was positively correlated to percent proteolyzed IGFBP-3 (r = 0.47, P = 0.0001) (not shown).
Variations in IGFBP-3 proteolysis and the IGF-I/intact IGFBP-3 ratio as related to nutritional status and indices of glucose homeostasis
Relationships with BMI. In healthy subjects, there seemed to
be an inverse correlation between BMI and percent proteolyzed IGFBP-3,
but it was not significant (r = -0.31, P = 0.11).
In IDDM patients, by contrast, there was a negative correlation between
these two parameters (r = -0.42, P = 0.008) (Fig. 5). In neither group of NIDDM patients
was there any such relationship: although approximately half of these
patients were obese, there was no difference in percent proteolyzed
IGFBP-3 between patients with BMI above and below 28.
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).
Because insulin requirements are greater in obese IDDM patients, owing
to the associated insulin resistance (18), a positive
relationship could be expected between BMI and the insulin dosage
administered. This proved to be true (r = 0.38, P
= 0.02, Fig. 5). The relationship was not significant in
insulin-treated NIDDM patients; but when all insulin-treated patients
were grouped together, a positive correlation emerged (r = 0.39,
P = 0.003, not shown). It therefore seemed possible
that the decrease in percent proteolyzed IGFBP-3 with BMI seen in IDDM
patients is related to the dosage of insulin received. However, we
observed no correlation between these two parameters, either among IDDM
patients or among insulin-treated NIDDM patients.
Relationships with blood glucose and HbA1c. No relationships were found between fasting glycemia and percent proteolyzed IGFBP-3 or IGF-I/intact IGFBP-3 ratio in the three groups of diabetics, whether considered separately or as a single group.
Among insulin-treated diabetics, there was a weak negative correlation
between percent HbA1c and percent proteolyzed IGFBP-3 (r = -0.25,
P = 0.05). The corollary of an inverse relationship
between percent HbA1c and IGF-I/intact IGFBP-3 ratio proved to be
highly significant (r = -0.33, P = 0.008, Fig. 6). There was no such relationship with
IGF-I levels.
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). In the latter, there was an inverse
relationship between IGF-I and IGFBP-1 levels (r = -0.47,
P = 0.01, not shown). In insulin-treated diabetics,
there was no relationship between IGFBP-1 levels and either IGF-I
levels or IGF-I/intact IGFBP-3 ratio.
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Relationship with IGF-I levels. The results shown in Fig. 2
indicated that, in at least half of the GH-deficient and acromegalic
patients, percent proteolyzed IGFBP-3 values were within the mean ±1
SD range of normals. This suggested an absence of
relationship with IGF-I levels. The lack of correlation between percent
proteolyzed IGFBP-3 and IGF-I proved true for these patients, normals,
and diabetics (r < 0.1 for each group) (Fig. 8). Detailed analysis of the data
revealed no difference between means for percent proteolyzed
IGFBP-3 in untreated acromegalics and patients in whom treatment
had restored IGF-I levels to within the normal range. In addition, in
12 GH-deficient patients investigated before and after GH treatment,
there was no significant change in percent proteolyzed IGFBP-3 values
(Table 2).
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): for
normal subjects, r = 0.65, P = 0.0001; for
GH-deficient patients, r = 0.59, P = 0.007; for
acromegalics, r = 0.37, P = 0.02; and for
diabetics, r = 0.40, P = 0.0001. The concurrent
increase in percentage of proteolyzed IGFBP-3 with increase in IGFBP-3
level suggests some mechanism regulating IGF-I bioavailability. The
corollary should therefore be a negative correlation between
IGF-I/total IGFBP-3 and percent proteolyzed IGFBP-3. This proved true
for normal subjects (r = -0.40, P = 0.02) and
diabetics (r = -0.30, P = 0.005) where the
correlation was significant, but less so for GH-deficient patients
where it was not (r = -0.33, P = 0.13) (Fig. 10). When data for GH-deficient
patients were grouped with those for normal subjects or diabetics, the
correlations became significant (r = -0.37, P =
0.007; and r = -0.29, P = 0.0001, respectively,
not shown). In acromegalics, there was no correlation between
IGF-I/total IGFBP-3 and percent proteolyzed IGFBP-3 (r = -0.21,
P = 0.25).
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Discussion |
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Role of IGFBP-3 proteolysis in IGF-I bioavailability
Age had little influence on percent proteolyzed IGFBP-3 in any of the groups of subjects tested, all of whom were adults; whereas in normals and diabetics, IGF-I (and to a lesser extent, IGFBP-3) levels declined significantly with age. This is a well-established physiological drop (17, 19) resulting from diminished GH secretion (16, 20). The outcome in older subjects is a reduced IGF-I/intact IGFBP-3 ratio and, hence, reduced IGF-I bioavailability that is not offset by increased IGFBP-3 proteolysis.
In almost all untreated GH-deficient patients, percent proteolyzed IGFBP-3 was in the normal or high normal range, and the IGF-I/intact IGFBP-3 ratios were within the normal range. In these patients then, the bioavailability of plasma IGF-I was not significantly enhanced by IGFBP-3 proteolysis, reflecting poor compensation for the defective IGF-I secretion. In acromegalics, such regulation seems to be absent, because the values for proteolyzed IGFBP-3 were also in the normal or high normal range, with a significantly elevated mean. The enlarged exchangeable fraction of IGF-I bound to IGFBP-3 would therefore aggravate the effects of excessive IGF-I synthesis induced by high GH secretion.
Among diabetics, percent proteolyzed IGFBP-3 was within the normal or high normal range, and the means were significantly higher for IDDM patients of less than 45 yr and insulin-treated NIDDM patients. Such increased IGFBP-3 proteolysis has been reported in untreated diabetes for adults with NIDDM (21) and children with IDDM (22). In our patients, the increased IGFBP-3 proteolysis meant that the IGF-I/intact IGFBP-3 ratio was maintained at a normal level in less-than-45-yr-old IDDM patients despite their significantly depressed IGF-I levels, and at a level above normal in the older IDDM patients and in NIDDM patients. The increased IGFBP-3 proteolysis can be interpreted as a regulatory mechanism mitigating the deficit in hepatic IGF-I synthesis that results from insulin deficiency and/or resistance (11, 23). The enhanced IGF-I bioavailability could reduce GH levels via its negative feedback effect on GH secretion, thus improving insulin sensitivity (24). It could also promote muscular glucose uptake, particularly because, in NIDDM patients, increased expression of insulin/IGF-I hybrid receptors, which have preferential affinity for IGF-I (25), has been observed.
The influence of nutritional status and glucose homeostasis on IGFBP-3 proteolysis was evident in insulin-treated diabetics, percent proteolyzed IGFBP-3, and the IGF-I/intact IGFBP-3 ratio being inversely related to BMI and percent HbA1c. When diabetes is associated with obesity, and hence insulin-resistance (18), or when there is poor glycemic control (as reflected by elevated HbA1c), the diminished IGFBP-3 proteolysis and IGF-I bioavailability can be considered as appropriate adaptation to limit energy use required for IGF-dependent cell processes.
A further index of this adaptation is the inverse relationship between IGF-I/intact IGFBP-3 ratios and IGFBP-1 levels. Insulin down-regulates hepatic synthesis of IGFBP-1 (26). Insulin deficiency provokes an increase in circulating IGFBP-1, which, through its ability to sequester free IGF-I (26), would join with the drop in IGF-I/intactIGFBP-3 ratio in diminishing IGF-I bioavailability.
An unexpected finding in this study was the positive correlation between total IGFBP-3 levels and percent proteolyzed IGFBP-3 in healthy adults and the three groups of patients. This would be physiologically significant, in that it implies an autoregulatory mechanism controlling the bioavailability of IGF-I bound to IGFBP-3 in the 140-kDa complexes. Such regulation is well illustrated by the negative correlation between IGF-I/total IGFBP-3 ratios and percent proteolyzed IGFBP-3 noted in all groups except the acromegalics (where the regulation seems to have been overcome). It is well known that hepatic synthesis of IGF-I and IGFBP-3 are closely coordinated (2); but in acromegalics, the capacity for GH-dependent synthesis of IGF-I surpasses that of IGFBP-3, the mean IGF-I/total IGFBP-3 ratio being 1.11, as opposed to approximately 0.4 for the other groups (results not shown).
Regulation of IGFBP-3 proteolysis
Regulation of IGFBP-3 proteolytic activity according to IGF-I/total IGFBP-3 ratio would necessitate controlled production of IGFBP-3 proteases or protease inhibitors. These, however, remain to be identified. Cation-dependent serine proteases are major contributors toward the IGFBP-3 proteolytic activity of pregnancy serum (27, 28), sera of NIDDM patients (21), and lymph (29). Plasmin acts as an IGFBP-3 protease in a variety of cell systems (30, 31). However, its wide spectrum of action and its poor specificity for the different IGFBPs would suggest that it plays a minor role in the degradation of circulating IGFBP-3.
As regards the protease inhibitors, their nature also remains a question and, in the context of this study, so do the roles played by GH and/or IGF-I and insulin in affecting their production. Insulin, GH, and IGF-I all play major roles in protein metabolism. Insulin seems to act primarily in the inhibition of proteolysis, whereas GH and IGF-I stimulate protein synthesis (32).
Two serine-protease inhibitors (serpins), SPI 2.1 and 2.2, are produced by rat hepatocytes and are dependent on GH (33, 34). No data exist as to their possible effects on IGFBP-3 proteases. Nevertheless, in the light of our results, it seems unlikely that GH and/or IGF-I are necessary or sufficient to control synthesis of IGFBP-3 protease inhibitors. In neither healthy subjects nor any group of patients was there any relationship between IGF-I levels and percent proteolyzed IGFBP-3. Furthermore, there was no significant difference in percent proteolyzed IGFBP-3, either between untreated and successfully treated acromegalics, or between hypopituitary patients before and after GH therapy. This is consistent with the results of a recent study of IGFBP-3 proteolysis using Western immunoblotting. No difference was detected between pre- and posttreatment samples from GH-deficient patients who were given GH or from acromegalics given octreotide (35).
Insulin may well be involved in the control of IGFBP-3 protease inhibitor synthesis. The plasminogen activator inhibitor, PAI-1, is stimulated by insulin in HepG-2 hepatic cells (36) and vasculoendothelial cells (37). Together with Kupfer cells, these comprise the primary source of IGFBP-3 in the bloodstream (38, 39). In obese subjects and NIDDM patients, hyperinsulinism is accompanied by an elevation of plasma PAI-1 levels (40). Other protease inhibitors may be influenced by insulin. IGFBP-3 proteolysis is intensified in untreated IDDM patients and reduced after treatment (22). In the IDDM patients in this study, the BMI-related drop in percent proteolyzed IGFBP-3 and increased insulin dosage administered would be coherent with insulin stimulation of IGFBP-3 protease inhibitor synthesis, but there was no direct link between percent proteolyzed IGFBP-3 and insulin dosage. The inverse relationship between percent proteolyzed IGFBP-3 and percent HbA1c would indicate that, in poorly controlled diabetes, the balance between IGFBP-3 proteases and inhibitors shifts in favor of inhibitors. In this situation of insulin insufficiency, other factors must contribute toward the synthesis of protease inhibitors.
Our results therefore reveal a loop in the regulation of IGFBP-3 proteolysis, which intensifies when plasma IGFBP-3 concentrations rise or the IGF-I/total IGFBP-3 ratio falls, and vice versa. Via this regulatory loop, IGF-I bioavailability is adjusted according to IGF-I and IGFBP-3 concentrations in the blood. Control of the synthesis of IGFBP-3 proteases and protease inhibitors seems to be a determining aspect, although the origin and nature of these inhibitors and the factors controlling them remain to be elucidated. With aging, there is no intensification of IGFBP-3 proteolysis to compensate for the reduced GH-related drop in IGF-I levels. In the pathological conditions studied here, modulation of IGF-I bioavailability by limited IGFBP-3 proteolysis contributes toward an appropriate adaptation to insulin deficiency and/or resistance but not to disturbances of GH secretion. Determination of the IGF-I/intact IGFBP-3 ratio could prove a useful tool as an index of the exchangeable fraction of IGFBP-3-bound (bioavailable) IGF-I.
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Acknowledgments |
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Footnotes |
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Received July 21, 2000.
Revised December 14, 2000.
Accepted January 29, 2001.
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References |
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T. B. Twickler, M. J. M. Cramer, G. M. Dallinga-Thie, M. J. Chapman, D. W. Erkelens, and H. P. F. Koppeschaar Adult-Onset Growth Hormone Deficiency: Relation of Postprandial Dyslipidemia to Premature Atherosclerosis J. Clin. Endocrinol. Metab., June 1, 2003; 88(6): 2479 - 2488. [Full Text] [PDF]
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 M. T.B. Twickler, M.-J. M. Cramer, H. P.F. Koppeschaar, J. Malik, T. Stulc, R. Ceska, E. Conti, F. Crea, F. Andreotti, A. Juul, et al. Unraveling Reaven's Syndrome X: Serum Insulin-Like Growth Factor-I and Cardiovascular Disease * Response Circulation, May 27, 2003; 107(20): e190 - 192. [Full Text] [PDF]
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M. Binoux Tools Now Exist to Measure Circulating IGF-Binding Protein-3 Proteolysis, to Study Its Regulation and Its Effects on IGF-I Bioavailability J. Clin. Endocrinol. Metab., October 1, 2001; 86(10): 5088 - 5089. [Full Text] [PDF]
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) and IGF-I/intact IGFBP-3
ratios (•) in untreated GH-deficient patients and acromegalics
(untreated or under treatment but with IGF-I levels above the normal
mean + 2 SD) and in healthy subjects and diabetics less
than 45 yr old. Horizontal bars indicate the means and,
for the healthy adults, the mean ± SD.
