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Bristol Royal Hospital for Sick Children (E.C.C.), Bristol, United Kingdom BS2 8BJ; Department of Pediatrics, Addenbrookes Hospital (C.L.A., A.W., D.B.D.), Cambridge, United Kingdom CB2 2QQ; Sheikh Rashid Laboratory, Radcliffe Infirmary (J.S.S.), Oxford, United Kingdom OX2 6HE; and Department of Surgery, Bristol Royal Infirmary (J.M.P.H.), Bristol, United Kingdom BS2 8HW; and Department of Paediatrics (T.C.), Royal Victoria Infirmary, Newcastle upon Tyne, United Kingdom NE1 4LP
Address all correspondence and requests for reprints to: Prof. D. B. Dunger, Department of Pediatrics, Box 116, Addenbrooke’s Hospital, Hills Road, Cambridge, United Kingdom CB2 2QQ. E-mail: dbd25{at}cam.ac.uk
Abstract
To determine the role of IGF-binding proteins in mediating the direct effects of recombinant human IGF-I on insulin requirements in type 1(insulin-dependent) diabetes mellitus, overnight changes in IGF-I, IGF-II, and IGF-binding protein-1, -2, and -3, collected under euglycemic conditions, were compared in nine subjects after double blind, randomized, sc administration of recombinant human IGF-I (40 µg/kg) or placebo at 1800 h. On both nights a somatostatin analog infusion (300 ng/kg·h) suppressed endogenous GH production, and three timed discrete GH pulses (total, 0.029 IU/kg·night) ensured identical GH levels.
After recombinant human IGF-I administration, IGF-I levels and the IGF-I/IGF-binding protein-3 ratio increased [mean ± SEM:IGF-I, 401 ± 22 ng/ml; placebo, 256 ± 20 ng/ml (P = 0.0002); IGF-I, 0.108 ± 0.006; placebo, 0.074 ± 0.004 (P = 0.0003), respectively], and insulin requirements decreased (IGF-I, 0.12 ± 0.03; placebo, 0.23 ± 0.03 U/kg·min; P = 0.008). The normal within-individual inverse relationships between insulin and IGF-binding protein-1 levels were observed (lag time 2 h: r = -0.34; P < 0.01). Yet despite reduced free insulin levels (8.5 ± 1.5; placebo, 12.2 ± 1.2 mU/liter; P = 0.03), IGF-binding protein-1 levels were reduced after recombinant human IGF-I administration (53.7 ± 6.8; placebo, 82.2 ± 11.8 ng/ml; P = 0.008). The largest reductions in free insulin levels after recombinant human IGF-I and thus putative improvement in insulin sensitivity occurred in subjects with the smallest increase in the plasma IGF-I/IGF-binding protein-3 ratio (r = 0.7; P = 0.03). Taken together, these data are consistent with the hypothesis that transcapillary movement of IGF-I (perhaps mediated by IGF-binding protein-1), out of the circulation facilitates altered insulin sensitivity. These data have important implications for risk-benefit assessment of recombinant human IGF-I therapy in type 1 diabetes mellitus.
ABNORMALITIES OF circulating levels of the GH/IGF/IGF-binding protein (IGFBP) axis are well documented in subjects with type 1 (insulin-dependent) diabetes mellitus and are strongly implicated in the deterioration in glycemic control commonly occurring in adolescence (1). These disturbances of the GH/IGF axis in type 1 diabetes mellitus can be explained by portal insulin deficiency and consequent down-regulation of hepatic GH receptors, resulting in reduced circulating IGF-I levels (1). This is supported by data showing reduced circulating levels of GH-binding protein, the extracellular portion of the GH receptor, in children with type 1 diabetes mellitus (2, 3). IGF-I bioactivity and bioavailability are also reduced (4) because of abnormalities of circulating IGFBP levels; with reduced IGFBP-3 levels (the principal IGF carrier) and increased IGFBP-1 levels (an inhibitory binding protein) (5). GH hypersecretion occurs secondary to reduced negative feedback from low circulating IGF-I and reduced IGF bioactivity (6, 7). The use of recombinant human IGF-I (rhIGF-I) has therefore been proposed as an adjunct to insulin therapy, and previous studies have demonstrated improved circulating IGF-I levels, reversal of GH hypersecretion, and improvements in glycemic control (8, 9, 10, 11), although the mechanisms of action and the long-term effects of rhIGF-I are not yet clear. It has been suggested that these potential increases in IGF bioavailability at the tissue level after rhIGF-I therapy could contribute to the development of microvascular complications in type 1 diabetes mellitus (12), and it is therefore essential that the complex effects of rhIGF-I therapy on the GH/IGF/IGFBP axis are established.
We have recently reported data demonstrating that rhIGF-I has direct effects on insulin requirements/sensitivity in adolescents with type 1 diabetes mellitus despite maintenance of GH levels after IGF-I administration (13). We now present further data from the same model that indicate a role for the IGFBPs and IGF-I bioavailability in mediating these effects.
Materials and Methods
Subjects
Nine adolescents/young adults with type 1 diabetes mellitus (two males and seven females; median age, 16.9 yr; age range, 12.4–21.9 yr) in late puberty (Tanner stage 4–5) were studied in a randomized, double blind, placebo-controlled study. Subjects had had type 1 diabetes mellitus for at least 4.5 yr and were otherwise healthy and nonobese (body mass index range, 19.4–28.2 kg/m2). All subjects were on standard multiple injection regimens (median insulin dose, 1.04 U/kg·d; range, 0.74–1.48). The glycosylated hemoglobin was 9.5% (7.9–14.1%; normal range, 4.3–6.1%). Stimulated C peptide was undetectable in three subjects and was between 0.01 and 0.09 pmol/liter in the remainder (blood glucose, >7 mmol/liter). None of the subjects had any evidence of retinopathy, proteinuria/persistent microalbuminuria, or hypertension. Pregnancy was excluded before each study. The study protocol was approved by the Central Oxford ethics committee, and written informed consent was obtained from the subjects and, where appropriate, their parents.
Study design
Intermediate-acting insulin was withdrawn at least 36 h before each study, and euglycemia was maintained with regular soluble insulin injections. The last insulin injection was given at 1200–1300 h on the day of admission, and subjects then had a standardized meal at 1730 h before fasting overnight.
Each subject was admitted on two occasions (see Fig. 1
for study protocol), when either
rhIGF-I (40 µg/kg; Pharmacia & Upjohn, Inc., Stockholm,
Sweden) or placebo was administered sc into the anterior aspect of the
left thigh at 1800 h. A euglycemic clamp (7 mmol/liter) was
maintained overnight using a continuous insulin infusion, the rate of
which was determined by 15-min blood glucose levels and a computer
program (14). During both study nights endogenous GH
secretion was suppressed with an octreotide infusion (300 ng/kg·h;
Sandostatin, Sandoz Pharmaceuticals Corp.,
Camberley, UK) between 1800–0800 h, and three identical 1-h GH pulses
(9.6 mU/kg·h; Genotropin, Pharmacia & Upjohn, Inc.) were infused between 2000–2100, 2300–2400, and
0200–0300 h.
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Assays
Samples making up a profile were analyzed in the same batch in duplicate. Whole blood glucose was determined at the bedside by the glucose oxidase method (YSI, Inc. analyzer, Clarendon Scientific Ltd., Farnborough, UK). GH samples were kept at room temperature until the profile was completed, then spun and separated, and the plasma was stored at -20 C until assayed using a standard diagnostic immunoradiometric assay kit (Netria, St. Barts Hospital, London, UK) and international reference standard 80/505. The detection limit was 0.5 mU/liter. The interassay coefficients of variation (CV) were 9.4%, 7.7%, and 10.5% at 3.5, 15.2, and 77.4 mU/liter, respectively; intraassay CVs were 8.0%, 2.0%, and 3.4% at 2.9, 14.3, and 69.4 mU/liter, respectively.
Free insulin levels were measured with a double antibody RIA (Guildhay Antisera Ltd., Guildford, UK) after precipitation with polyethylene glycol (mol wt, 6000; Sigma, Poole, UK). Inter- and intraassay CVs were 5.5% and 8.6% at 12.2 and 47.2 mU/liter, and 2.6%, 4.6%, and 5.9% at 19.4, 38.3, and 54.9 mU/liter, respectively.
IGF-I levels were measured by RIA after acid-ethanol extraction. Assay sensitivity was 14 ng/ml. Intra- and interassay CVs were 11.3%, 6.5%, and 4.7% at 46, 246, and 706 ng/ml, and 10.5%, 12.1%, and 5.1% at 76, 198, and 706 ng/ml, respectively.
IGF-II levels were measured by RIA after removal of binding protein by formic acid/acetone. The intra- and interassay CVs were 6.5% and 7.6% at 874 ng/ml respectively, with cross-reactivities with IGF-I and insulin of 3.8% and less than 0.01%, respectively.
IGFBP-1 was measured by RIA using antisera and purified antigen provided by Dr. H. Bohn (Behringwerke, Marburg, Germany). Assay detection limit was 6 µg/liter. The interassay CVs were 10.3% and 9.1% at 9 and 353 µg/liter, respectively; intraassay CVs were 10.6% and 7.0% at 106 and 253 ng/ml, respectively.
IGFBP-2 was assayed using a double antibody RIA (Diagnostic Systems Laboratories, Inc., Webster, TX). The intraassay CVs were 8.5%, 6.2%, and 4.7% at 13.0, 32.2, and 94.4 ng/ml, respectively. The interassay CVs were 7.4%, 4.5%, and 7.2% at 2.7, 13.2, and 69.7 ng/ml, respectively.
IGFBP-3 levels were determined by RIA using recombinant glycosylated IGFBP-3 provided by Celtrix Pharmaceuticals, Inc. (Santa Clara, CA), for both standards and tracer. The normal range of the assay was 4160–6770 ng/ml. At 5000 ng/ml, the intra- and interassay CVs were 4.38% and 5.14%, respectively.
Plasma glucagon was measured by RIA using guinea pig antihuman glucagon antibodies for pancreatic glucagon, [125I]glucagon as tracer and glucagon standards, with reagents supplied by Linco Research, Inc. (St. Charles, MO). Inter- and intraassay CVs were 5–10%.
Analyses
Results are presented as the mean ± SEM. The overnight period (0400–0800 h) was defined as the steady state period when there was no significant change in blood glucose level with time as determined by ANOVA, nor any difference in mean plasma glucose levels between the two study nights; this period was used to calculate insulin levels as an index of insulin sensitivity.
Distributions of the data were examined for normality using the Kolmogorov-Smirnov goodness of fit test. Comparisons between means from the 2 study nights were made using t tests for paired samples. Comparisons between profiles from the 2 study nights were made using ANOVA for repeated measures.
Cross-correlation was used to determine the interrelationships between different hormones (15). This iterative technique establishes whether there are statistically coincident recurring waveforms (of any shape) within a data array. One data array is serially correlated against another with progressive step changes in the time relationship between the data. The result is dependent on both the relative amplitude of the waves or pulses (i.e. whether large pulses of one array are associated with large pulses in the second) and on the phase relationship between the arrays. It is not dependent on the absolute hormone concentrations or the regularity or irregularity of the waveforms. The correlation coefficient is at its greatest at a time lag equal to the mean phase difference between the hormone profiles. Cross-correlation yields an unbiased estimate of this phase difference and an assessment of statistical significance. The correlations can be pooled (using Fisher’s z transformation) to obtain an estimate of the overall likelihood of the significance and phase relationship of all available data.
Results
None of the subjects had a blood glucose level below 3 mmol/liter or symptoms of hypoglycemia on either study night. During the glucose steady state period (0400–0800 h) there was no significant change in glucose levels with time on either night and, furthermore, no significant difference in glucose levels after rhIGF-I (7.3 ± 0.3; placebo, 7.5 ± 0.3 mmol/liter; P = 0.4). There was no difference in fasting (0800 h) glucagon levels after rhIGF-I (47.9 ± 5.6; placebo, 48.5 ± 3.1 pg/ml; P = 0.7) or in steady state glucagon levels (53.2 ± 4.1; placebo, 52.0 ± 4.4 pg/ml; P = 0.5). As previously reported, there were significant reductions in overnight insulin infusion rates after rhIGF-I (0.12 ± 0.03; placebo, 0.23 ± 0.03 mU/kg·min; P = 0.015) and also in free insulin levels (8.5 ± 1.5; placebo, 12.2 ± 1.2 mU/liter; P = 0.02), largely related to the absence of the GH pulse-related changes in insulin requirements (13).
rhIGF-I administration resulted in sustained increases in IGF-I levels
(Fig. 2A) with higher peak IGF-I
levels (460.4 ± 26.9 ng/ml at 2300 h after rhIGF-I
vs. 302.2 ± 19.1 ng/ml at 2000 h after placebo)
and also higher steady state IGF-I levels (395 ± 23; placebo,
255 ± 20 ng/ml; P < 0.001). IGF-II levels (Fig. 2B) were significantly reduced after rhIGF-I (401.7 ± 32;
placebo, 481.2 ± 45 ng/ml; P = 0.014). Combined
levels of IGF-I plus IGF-II were elevated during the early part of the
night after rhIGF-I, but were not different during the steady state
period. Thus, there was a significant difference in the pattern of
change with time for combined IGF-I plus IGF-II levels (by ANOVA for
repeated measures, P = 0.03; Fig. 2C), and peak levels
of combined IGF-I plus IGF-II (2200 h) were increased after rhIGF-I
(122 ± 2.4; placebo, 103 ± 2.7 nmol/liter;
P < 0.01).
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, D and E, F). The
expected inverse relationship between insulin and IGFBP-1 was present,
as demonstrated by a reduction in IGFBP-1 levels on both study nights
after the start of the insulin infusion; on cross-correlation the
closest relationship was seen when IGFBP-1 levels were lagged by 2
h against free insulin levels (r = -0.34; P <
0.01). Nevertheless, despite the reductions in both circulating free
insulin levels and insulin infusion requirements after rhIGF-I
administration, steady state IGFBP-1 levels were significantly reduced
after rhIGF-I (78.2 ± 8.6; placebo, 57.5 ± 7.4 ng/ml;
P = 0.001; Fig. 2D). This difference was maximal at
10 h after the start of the insulin infusion; IGFBP-1 levels at
0300 h were 50.3 ± 10.6 ng/ml after rhIGF-I vs.
95.7 ± 18 ng/ml after placebo (P = 0.006). ANOVA
for repeated measures over the whole study period identified a
significant reduction in IGFBP-1 levels after rhIGF-I
administration (P = 0.018) and a significant change
with time (P < 0.001) on both nights, but no
alteration in the pattern of change after rhIGF-I (Fig. 2D). During the
steady state period, there were significant correlations between mean
IGFBP-1 levels and mean levels of IGF-I, IGF-II, and IGFBP-3 on both
the rhIGF-I and placebo nights (Table 1).
However, there were no apparent relationships between the change in
mean IGFBP-1 levels after rhIGF-1 or placebo and changes in IGF-I,
IGF-II, free insulin levels, or insulin infusion requirements.
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). Steady state
IGFBP-3 levels were also not changed after rhIGF-I administration, but
there was a significant difference in the variation of IGFBP-3 with
time between the 2 study nights (Fig. 2F; by ANOVA for repeated
measures:, P < 0.0001). IGFBP-3 levels rose after
rhIGF-I administration, with maximal divergence after 8 h; IGFBP-3
levels at 2300 h were 4100 ± 270 ng/ml after rhIGF-I
vs. 3600 ± 290 ng/ml after placebo (P
= 0.01). IGFBP-3 levels subsequently converged.
The steady state IGF-I/IGFBP-3 ratio (a measure of IGF bioavailability)
increased after rhIGF-I (0.34 ± 0.02; placebo, 0.23 ± 0.01;
P < 0.001; Fig. 3A).
Changes in the IGF-I/IGFBP-3 ratio were significantly related to
changes in insulin levels between the 2 nights. After rhIGF-I
administration, the largest reduction in circulating free insulin
levels was seen in subjects with the least increase in IGF-I/IGFBP-3
ratio (r = 0.7; P = 0.03; Fig. 3B), indicating
that the greatest reductions in free insulin levels were seen in
subjects with the least retention of bioavailable IGF-I in their
circulation.
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The potential role of rhIGF-I as an adjunct to insulin therapy in subjects with type 1 diabetes mellitus raises exciting possibilities, as studies have demonstrated a reduction in insulin requirement for euglycemia and improved glycemic control in both short- and long-term studies (8, 11, 13, 16, 17). Further studies are required to investigate long-term safety and efficacy; in particular, a greater understanding of its mechanisms of action are required. Correction of abnormalities in circulating IGF-I levels in type 1 diabetes mellitus by rhIGF-I administration could influence insulin requirements either by a negative feedback effect on GH hypersecretion (9) or by direct effects at the tissue level. Direct tissue effects, related to changes in IGF bioavailability, may, in turn, influence the development of long-term microvascular complications in type 1 diabetes mellitus (18).
In this study we compared the effects of a single sc injection of rhIGF-I with placebo on circulating levels of the IGFs and their binding proteins in a model in which GH pulsatility was the same on both study nights. The observed changes are therefore due to rhIGF-I, rather than being secondary to the negative feedback of IGF-I on circulating GH levels. We previously reported that these studies demonstrated a reduction in overnight insulin infusion requirements after rhIGF-I administration, with abolition of GH pulse-related changes in insulin requirements suggesting a direct effect of rhIGF-I on GH-mediated changes in insulin sensitivity (13). We now describe the complex changes seen in circulating IGFs and their binding protein levels after rhIGF-I administration.
IGF-I levels were restored to the normal range (13), with an increase of 150%, but IGF-II levels were reduced to 80% of placebo night levels, resulting in a modest change in overall circulating IGF levels, in agreement with a previous study of rhIGF-I administration in type 1 diabetes mellitus (19). IGF-II reduction was most marked at 6 h after rhIGF-I administration; this could result from either movement of IGF-II out of the circulation into the tissues in response to acute changes in circulating IGF-I levels or decreased production of IGF-II. In vitro studies indicate that IGF-II can alter insulin sensitivity in both human and animal muscles within the physiological range (20, 21), but as the full physiological role of IGF-II is not clear, we can only speculate as to whether these changes in circulating levels of IGF-II have any biological importance. The changes may simply reflect the mechanisms by which potentially free IGF-I is assimilated into the bound IGF pool, and the IGF-II is displaced from the binding protein complexes and rapidly degraded.
The changes in circulating IGFBP-1, -2, and -3, however, were not consistent with simply "mopping up" spare free IGF-I. Levels of IGFBP-3 increased transiently, whereas levels of IGFBP-1 actually decreased relative to those on the placebo night, and IGFBP-2 levels did not change during the study period. IGFBP-2 levels have been shown to be regulated by insulin and GH independently (22, 23, 24). Increases were noted after rhIGF-I infusion in normal adults (22) and by day 7 of chronic administration of rhIGF-I in children with type 1 diabetes mellitus (19). In both of these studies rhIGF-I administration suppressed endogenous GH and insulin levels (19). In our study, however, in which rhIGF-I and GH were given concomitantly to subjects with type 1 diabetes mellitus, IGFBP-2 levels were not changed acutely. IGFBP-3 is regulated principally by GH, but it is probably also altered by IGF-I levels, as infusions of IGF-I induced changes in IGFBP-3 levels in hypophysectomized or diabetic rats (25, 26). We identified a transient rise in overnight IGFBP-3 levels after rhIGF-I administration, which was maximal at 8 h, but subsequently decreased. Thrailkill et al. (19) did not sample overnight, but found no change in IGFBP-3 levels measured 2–4 h post-IGF-I injection. We also recently demonstrated transient changes in IGFBP-3 levels even when GH levels were reduced after rhIGF-I administration in subjects with type 1 diabetes mellitus (27).
It has been proposed that IGFBP-1 has an active role in glucose homeostasis, linking the IGF system to carbohydrate intake and nutritional status (28, 29, 30). IGFBP-1 levels are regulated principally by insulin and to a lesser extent by glucose levels, but conflicting data have emerged about changes after rhIGF-I administration. In our study IGFBP-1 levels were decreased after rhIGF-I despite the significant reduction in circulating insulin levels. The expected negative correlation between insulin and IGFBP-1 was still evident, as demonstrated by cross-correlation analysis linking insulin with IGFBP-1 levels with a lag time of 2 h. Decreased IGFBP-1 levels were reported in an earlier study involving both rhIGF-I and GH administration to subjects with type 1 diabetes mellitus (13), but increased IGFBP-1 levels were observed after rhIGF-I infusion alone in four adolescent type 1 diabetes mellitus subjects (31). In normal subjects IGFBP-I levels increased after rhIGF-I administration (32). These discrepant results may be due to differences in rhIGF-I dose, timing of venous sampling, or different study designs and therefore varying effects on circulating GH and insulin levels. Other factors may also influence IGFBP-1 levels. In vitro studies have shown that glucagon increases IGFBP-1 secretion (33), although this effect has not been demonstrated in vivo. Octreotide may cause an increase in IGFBP-1 levels, as reported in patients with acromegaly (34). In the current study, however, glucagon levels were equally suppressed on both study nights by an identical octreotide infusion and therefore cannot account for any differences between study nights.
In addition to reducing IGFBP-1 mRNA expression, insulin may reduce circulating IGFBP-1 levels by altering transcapillary movement of IGFBP-1 out of the circulation (35). Our observations indicate that rhIGF-I may have similar effects on reducing circulating IGFBP-1 levels. The pharmacokinetic profiles of both IGFBP-1 and IGFBP-2 suggest that they equilibrate with the extravascular compartment and may play a role in transcapillary transport of IGF-I (36) and thus IGF-I tissue availability. In support of this hypothesis, that transcapillary movement of IGF-I (perhaps mediated by IGFBP-1) is linked to its bioactivity, we observed an inverse relationship between changes in insulin levels required to maintain euglycemia and the IGF-I/IGFBP-3 ratio. A higher IGF-I/IGFBP-3 ratio, reflecting retention of bioactive IGF-I in the circulation, is associated with minimal changes in insulin requirements.
This hypothesis suggests that it is IGF bioactivity in the tissues, not the circulation, that is important for the observed effects on insulin sensitivity, and this is important in the context of the development of microvascular complications in type 1 diabetes mellitus. Several studies have linked the development of diabetic nephropathy or neuropathy with tissue-specific abnormalities in IGF levels or changes in IGF bioavailability (37, 38, 39). A deterioration in retinopathy has been described after rhIGF-I administration to subjects with type 1 diabetes mellitus given doses greater than 80 µg/kg (40). This may have been a transient phenomenon related to rapid improvement in glycemic control (41), or alternatively, it could have been related to the changes in IGFBP-1 and IGFBP-2 observed in that 6-wk study. In the study reported by Acerini et al. (11) using a lower dose of 40 µg/kg·d, no significant changes in IGFBPs were observed, and retinopathy scores were unchanged after 6 months. Thus, changes in ratios of bound and unbound IGF-I may be critical to the risk of aggravation of complications after rhIGF-I administration.
In conclusion, we have described the changes after a single sc injection of rhIGF-I to subjects with type 1 diabetes mellitus, which restored IGF-I levels to the normal range, whereas GH levels were maintained by exogenous infusions. Complex changes in the IGF/IGFBP system occurred that could be explained by movement of IGF-I into the tissues and subsequent alterations in insulin sensitivity. Additional studies are required to clarify the potential role of rhIGF-I therapy in type 1 diabetes mellitus, investigating its long-term effects on glycemic control and the systemic and tissue level changes in the IGF/IGFBP system, which may determine the role of IGF-I in the pathogenesis of microangiopathic complications of type 1 diabetes mellitus.
Acknowledgments
We thank Pharmacia & Upjohn, Inc., for providing the rhIGF-I used in this study, and Paul Griffiths for statistical advice. The glucagon assays were performed at the RIA Core Facility of the Diabetes Research and Training Center of Washington University School of Medicine (St. Louis, MO). The IGFBP-2 assays were performed by J. Jones at the Institute of Child Health Laboratories (London, UK).
Footnotes
This work was supported by Pharmacia & Upjohn, Inc. (Stockholm, Sweden; to E.C.C.) and the Wellcome Foundation (to J.S.S.).
Abbreviations: CV, Coefficient of variation; IGFBP, IGF-binding protein; rhIGF, recombinant human IGF.
Received January 19, 2001.
Accepted April 6, 2001.
References
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)
at 1800 h in nine adolescents with type 1 diabetes mellitus during
euglycemic clamp studies involving continuous insulin and somatostatin
analog infusions and three 60-min exogenous GH pulses at 2000, 2300,
and 0200 h.