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Original Studies |
Department of Internal Medicine, Erasmus University, Rotterdam, The Netherlands
Address all correspondence and requests for reprints to: Dr. Jamjl Janssen, Department of Internal Medicine III, Room D438, University Hospital Dijkzigt, Dr. Molewaterplein 40, 3015 GD Rotterdam, The Netherlands.
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Abstract |
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Serum free and total IGF-I, IGF-binding protein-1 (IGFBP-1), and IGFBP-3 levels were measured in 56 insulin-treated IDDM patients and 52 healthy sex- and age-matched controls. Diabetic retinopathy was established by direct fundoscopy. In 54 IDDM patients, the glomerular filtration rate (GFR) and effective renal plasma flow were calculated from the clearance rate of [125I]iothalamate and [131I]iodohippurate sodium.
Fasting free IGF-I, total IGF-I, and IGFBP-3 levels were significantly lower in IDDM patients than in age- and sex-matched healthy controls (free IGF-I, P < 0.005; total IGF-I, P < 0.001; IGFBP-3, P = 0.001), whereas IGFBP-1 levels were higher (P < 0.001).
In IDDM subjects, decreases in free IGF-I, total IGF-I, and IGFBP-3 levels with age were observed (free IGF-I, r = -0.27 and P = 0.05; total IGF-I, r = -0.52 and P < 0.001; IGFBP-3, r = -0.37 and P = 0.005). Free IGF-I was inversely related to fasting glucose in IDDM subjects (r = -0.35; P = 0.01), whereas the relationship between total IGF-I and fasting glucose did not reach significance (r = -0.27; P = 0.06).
Age-adjusted free IGF-I levels were significantly higher (P < 0.05) in IDDM subjects with retinopathy than in subjects without retinopathy after adjustment for age.
Total IGF-I and IGFBP-3 levels were positively related to GFR (total IGF-I, r = 0.35 and P < 0.05; IGFBP-3, r = 0.28 and P < 0.05). Both of these differences lost significance after adjustment for age.
Free IGF-I, total IGF-I, and IGFBP-3 levels were lower and IGFBP-1 levels were higher in insulin-treated IDDM subjects compared to those in age- and sex-matched controls. Free IGF-I, total IGF-I, and IGFBP-3 levels decreased significantly with age in IDDM subjects. Age-adjusted free IGF-I levels in subjects with diabetic retinopathy were higher than those in subjects without diabetic retinopathy. Total IGF-I and IGFBP-3 levels were positively related to GFR in IDDM subjects, but these relations were lost after adjustment for age. Measurement of serum free IGF-I levels in IDDM subjects did not have clear advantages compared to that of total IGF-I, IGFBP-1, and IGFBP-3 levels. Serum IGF-I and IGFBPs reflect their tissue concentrations to a various degree. Consequently, extrapolations concerning the pathogenetic role of the IGF/IGFBP system in the development of diabetic complications at the tissue level remain speculative.
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Introduction |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Blood-borne IGF-I may also contribute to the glomerular hyperfiltration observed in early diabetic nephropathy (6). However, circulating levels of total IGF-I do not closely correlate to renal hypertrophy, and there is a lack of correlation between kidney size and the progressive decline in the glomerular filtration rate (GFR) in diabetic patients, suggesting that factors important for growth might not necessarily be involved in renal hemodynamics (7, 8).
Part of the controversy concerning serum IGF-I levels in IDDM may be explained by methodological problems in the IGF-I assay. Until recently, the assay methods used to measure IGF-I in serum were not able to distinguish among unbound, free IGF-I, and IGF-I bound to binding proteins (IGFBPs) (9). IGFBP-3 appears to be the primary regulator of IGF levels in response to changes in circulating GH levels and serves as a storage pool for IGF-I (10), whereas IGFBP-1 appears to be the primary regulator of IGF-I levels in response to changes in circulating insulin levels (11).
Determination of free IGF-I instead of total IGF-I might be a way to gain insight into the concentrations and the role of the IGF-I/IGFBP system in IDDM (7). Free IGF-I, by analogy with sex and adrenal steroids and thyroid hormones, may be the major biologically active, hormonal form of IGF-I (12).
Here we report the results of a study in which we investigated the relationships of circulating free IGF-I, total IGF-I, IGFBP-1, and IGFBP-3 levels to GH release and to the presence of diabetic retinopathy and renal hemodynamics in patients with IDDM.
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Subjects and Methods |
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The study group consisted of 56 consecutive patients with IDDM who visited our out-patient clinic. Inclusion criteria were IDDM, age 18 yr or older at the time of the study, age at diagnosis below 40 yr and insulin dependency within 6 months of diagnosis, no other medication than insulin, glycosylated hemoglobin (HbA1c) less than 11%, normal serum creatinine (50–110 µmol/L), and no overt nephropathy (albuminuria, <300 mg/24 h in 2 of 3 measurements). About half of these patients were part of a previous study in which the dynamics of GH secretion were studied (13). All patients were on an insulin dose schedule of 2–4 times daily, using combinations of short-acting and intermediate long-acting insulin. Fifty-two age- and sex-matched controls without evidence of disease were recruited from the normal urban population and staff members of our hospital to measure free and total IGF-I levels. The study was approved by the hospital ethical review committee, and all patients and volunteers gave their informed consent.
Blood sampling
Blood was collected between 0800–0900 h in the morning after an overnight fast in both IDDM patients as well as the control group. The last insulin injection on the day before blood sampling was administered at 2200 h by IDDM subjects receiving intensive insulin therapy (four daily injections, injection of an intermediate long-acting insulin at 2200 h) and at 1800 h by IDDM subjects receiving conventional therapy (two daily injections of a mixture of short- and medium-acting insulins).
Hemodynamic and renal function tests
In 54 of 56 subjects with IDDM, renal and hemodynamic function tests were performed. The GFR and effective renal plasma flow (ERPF) were calculated from the clearance of [125I]iothalamate and [131I]iodohippurate sodium, respectively, as described in detail previously (14). Blood glucose measurements were performed every 30 min. Glucose (50 mg/kg·h) and insulin (daily dose in units divided by 100/h) was given iv. To maintain the blood glucose concentration between 6.0–10 mmol/L, the rate of glucose infusion was adjusted according to the blood glucose levels. The mean GFR in a group of normal subjects (n = 18, 10 men and 8 women; mean age, 39 yr; range, 25–61 yr) was 107 (range, 88–130) mL/min·1.73 m2. A GFR greater than 130 mL/min·1.73 m2 indicated hyperfiltration.
Blood pressure was measured every 5 min during a 60-min period in the supine position using an automatic device (Datascope, Accutor 1a, Parasmus, NJ). The mean of at least six values between 30–60 min was calculated.
Analytical methods
The free IGF-I immunoradiometric assay (Diagnostics System Laboratories, Webster, TX) used in the present study needs no sample extraction. The assay has been previously described (15, 16). In short, serum samples (100 µL) or recombinant human IGF-I standards were added to tubes containing a dense coating of the primary high affinity IGF-I antibody and incubated at 2 h at room temperature. No residual IGFBP-1 or IGFBP-3 was detectable after the first wash according to the manufacturer’s data (15). The tubes were washed and incubated for 2 h at room temperature with the secondary radiolabeled antibody, washed three times with deionized water, and counted. The lower limit of detection is 4 pmol/L. The cross-reactivity with IGF-II is less than 0.01%. The intra- and interassay coefficients of variation (CVs) for the free IGF-I assay are 10.3% and 10.7%, respectively, at a plasma level of 0.01 nmol/L (n = 8).
Total IGF-I was determined by a commercially available RIA (Medgenix Diagnostics, Brussels, Belgium; intra- and interassay CVs, 6.1% and 9.9%). Immunoradiometric assays were used for the measurement of IGFBP-1 and IGFBP-3 (Diagnostics System Laboratories; intra- and interassay CVs for IGFBP-1, 5.2% and 6.0%; intra- and interassay CVs for IGFBP-3, 0.56% and 1.9%). HbA1c was measured by high performance liquid chromatography (normal range, 5.0–6.3%; Variant HPLC-Bio-Rad, Veenendaal, The Netherlands), and blood glucose concentrations were measured in venous whole blood by an automatic hexokinase method (Boehringer Mannheim, Mannheim, Germany). Urinary albumin was measured with a radial immunodiffusion on agarose gel that contained 0.4 µL antialbumin antiserum (Dako A 001, Dakopatts, Copenhagen, Denmark) (17). The lower limit of detection was 2 mg/L, and the interassay variability was 8%. The means of the results of three collection periods are presented.
Diabetic retinopathy
Diabetic retinopathy (DRP) was established by direct fundoscopy, as judged by an experienced ophthalmologist, and was graded as no DRP, background DRP, or proliferative DRP (18).
Statistical analysis
Baseline clinical characteristics are presented as the mean and
range. Free IGF-I, total IGF-I, IGFBP-1, IGFBP-3, and urinary albumin
excretion have a log normal distribution and are, therefore, presented
as the geometric mean. IDDM subjects were compared with healthy
controls. For the healthy control group we did not match on an
individual basis, but performed frequency matching, i.e.
selecting our controls in such a way that age and sex distribution were
similar in cases and controls. For diabetic subgroups, mean and 95%
confidence intervals are presented. Baseline differences between
variables were analyzed with one-way ANOVA. Pearson’s partial
correlation coefficients were calculated to analyze the associations
between variables and to test significance. Free IGF-I, total IGF-I,
IGFBP-1, and IGFBP-3 did not meet the criteria for normality and were
logarithmically transformed before analysis. After this transformation,
a normalization of the distribution was achieved. The results presented
in Table 3
were adjusted for age using linear regression analysis with
(total and free) IGF-I, IGFBP-1, and IGFBP-3 as dependent variables and
age and retinopathy (yes/no) as independent variables. The relations
between total IGF-I and IGFBP-3, respectively, and GFR were adjusted
for age using linear regression analysis with total IGF-I and IGFBP-3
as dependent variables and age and GFR as independent variables.
Two-sided P < 0.05 was considered significant. All
statistical analyses were performed with Stata statistical package
(Computing Resource Center, Santa Monica, CA).
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Results |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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The baseline clinical characteristics of 56 IDDM subjects (23
women and 33 men) are presented in Table 1. In the 52
sex- and age-matched controls, the female/male ratio was 21/31 and not
significantly different from that in the IDDM patients. The mean age in
the control group was 34.5 yr (SD, 13.8; range, 18–64) and
did not differ from that in the IDDM group. Twenty-two of the 54 IDDM
patients had a GFR above 130 mL/min·1.73 m2 and were
classified as hyperfiltrators.
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and Table 2.
Serum free and total IGF-I levels were significantly lower in the IDDM
patients than in the control group. The ratio between free IGF-I and
total IGF-I levels was lower, but not statistically different, between
IDDM patients and healthy controls. Serum IGFBP-3 levels were
significantly lower and IGFBP-1 significantly higher in the IDDM
patients than in the control group (Fig. 1 and Table 2).
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Both free IGF-I as well as IGFBP-3 levels decreased significantly with
age in diabetic patients, whereas IGFBP-1 levels did not change with
age (Fig. 2, B and C). In the normal individuals such
decreases in free IGF-I and IGFBP-3 with age were not statistically
significant (Fig. 2, B and C), whereas total IGF-I levels were
inversely related to age in both IDDM and healthy controls (Fig. 2A).
Free IGF-I and IGFBP-3 levels tended to be disproportionately lower in
IDDM than healthy controls with increasing age (Fig. 2, B and C).
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). The
relation between total IGF-I and fasting glucose did not reach
significance (r = -0.27; P = 0.06), and fasting
IGFBP-1 levels were positively related to fasting glucose levels in
IDDM subjects (Fig. 3). Fasting glucose levels were positively related
to HbA1c (r = 0.36; P =
0.01).
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The 27 IDDM patients with retinopathy were older than the 29
patients without retinopathy, the duration of diabetes was longer,
blood pressure was higher, and the GFR and ERPF were lower (Table 3
). Free and total IGF-I, IGFBP-1, IGFBP-3,
HbA1c and fasting glucose levels were not
different between the 2 groups. When adjusted for age, the patients
with retinopathy tended to have higher free IGF-I levels
(P = 0.04).
Renal hemodynamics
Using 130 mL/min·1.73 m2 as a cut-off, 22 patients
had hyperfiltration, and 32 had a normal GFR (Table 4).
The patients with hyperfiltration were younger, and blood pressure was
not different in the two groups. In IDDM subjects, GFR decreased
significantly with age (r = -0.53; P < 0.001).
Free IGF-I, IGFBP-1, and IGFBP-3 levels tended to be higher in the IDDM
patients with hyperfiltration than in subjects with a GFR below 130
mL/min·1.73 m2 (Table 4), but the differences were not
significant.
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After adjustment for fasting glucose, IGFBP-1 levels were inversely related to GFR (r = -0.30; P < 0.05). Total and free IGF-I levels were not related to ERPF.
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Discussion |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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We observed a significant decrease in free IGF-I and IGFBP-3 levels during aging in IDDM subjects, but not in our healthy control population. Therefore, free IGF-I and IGFBP-3 levels tended to be disproportionately lower in IDDM than in our healthy controls. A difference between IDDM subjects and healthy controls was not observed for total IGF-I levels with increasing age. In accordance with this, Graubert et al. reported that in the streptozocin-induced diabetic rat, serum free IGF-I concentrations were more depressed than total IGF-I (23). Reduced free IGF-I concentrations probably account for the reduced IGF-I bioactivity in diabetic serum, as reported in earlier studies (24).
Free IGF-I levels were inversely related to fasting glucose levels in IDDM subjects, whereas fasting glucose levels were positively related to HbA1c. IGF-I exerts a tonic hypoglycemic effect in the circulation (25), and decreased IGF-I levels may induce an increase in serum glucose levels. As we observed a significant decrease in free IGF-I levels during aging in IDDM subjects, this decrease might contribute to gradually higher serum glucose levels and thereby to the development of diabetic microvascular complications (26).
IGFBP-1 levels were positively related to fasting glucose levels. A previous study in IDDM subjects also reported a positive relationship between IGFBP-1 and fasting plasma glucose levels (27). IGFBP-1 administration causes an acute rapid increase in plasma glucose levels (25), which suggests that increased IGFBP-1 levels result in decreased availability of free IGF-I levels (28). IGFBP-1 is thought to modulate the free fraction of IGF-I. The inverse relationship between free IGF-I and IGFBP-1 in our study supports the hypothesis that circulating free IGF-I levels are low(er) when IGFBP-1 levels are high (15).
Free IGF-I levels tended to be higher in IDDM patients with retinopathy after adjustment for age. However, this result needs to be interpreted with caution because retinopathy scoring was performed with direct ophthalmoscopy, a technique that may result in some misclassification of subjects and could, therefore, materially alter the results and the conclusions of our study.
An increased GFR is considered an early symptom in diabetes mellitus (29). We observed lower values for total serum IGF-I in IDDM patients than in sex- and age-matched controls, which suggests at first glance no important role for IGF-I in the pathogenesis of hyperfiltration in IDDM. Nevertheless, in our study total IGF-I levels were positively related to the GFR. The observed correlation coefficient between total IGF-I and GFR is in agreement with previous data showing that IGF-I leads to an augmentation of GFR by 10–20% (30, 31). In an experimental animal model of diabetes that was characterized by increased GFR, increased binding of IGF-I to its renal IGF-I receptors was found compared to that in controls (32). Increased binding of IGF-I to the IGF-I receptor in the IDDM subjects despite low serum total IGF-I levels might also explain the observed relationship between total IGF-I and GFR in our study. However, this relationship was lost after adjustment for age, which suggests that the relationship between GFR and serum total IGF-I levels is age dependent and lost after the physiological age-dependent decrease in serum total IGF-I levels with aging.
In conclusion, free and total IGF-I and IGFBP-3 levels are lower and IGFBP-1 levels are higher in IDDM subjects than in controls. Free IGF-I and IGFBP-3 levels decrease significantly with age in IDDM subjects between 20–65 yr of age. Higher (age-adjusted) free IGF-I levels were observed in IDDM subjects with diabetic retinopathy than in subjects without retinopathy, but this difference was lost after further adjustment for glucose levels. Total IGF-I and IGFBP-3 levels were related to GFR in IDDM subjects, but this relation was lost after adjustment for age.
Measurement of serum free IGF-I levels in IDDM subjects did not provide clear advantages compared to that of total IGF-I, IGFBP-1, and IGFBP-3 levels. Serum IGF-I and IGFBPs reflect their tissue concentrations to various degrees. Consequently, extrapolations concerning the pathogenetic role of the IGF-IGFBP system in the development of diabetic complications at the tissue level remain speculative.
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Footnotes |
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Received January 7, 1997.
Revised April 17, 1997.
Accepted May 13, 1997.
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References |
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