IGFs (insulin-like growth factors), which in an unbound form induce glucose and amino acid uptake, circulate bound to IGFBPs (IGF-binding proteins), which modulate their bioavailability and activity. The aim of the present study was to examine the effect of a standard meal [2301 kJ (550 kcal)] on the serum levels of IGFBP-1 in obese patients with T2DM (Type 2 diabetes mellitus), non-obese patients with T1DM (Type 1 diabetes mellitus) and healthy controls, using the artificial pancreas (Biostator®) to obtain a normal glycaemic response to the meal. IGFBP-1 levels decreased by 50% over 2 h following the meal at a similar clearance in both the healthy controls and patients with T1DM, but no significant decline was seen in the patients with T2DM, despite a several-fold increase in insulin levels. The patients with T2DM were also studied during Sandostatin® (somatostatin) infusion to decrease the inappropriate secretion of glucagon during the meal. During the 210 min of somatostatin infusion, the glucagon response was suppressed and IGFBP-1 levels were increased concomitantly with the peak in insulin levels, without any significant decrease after the meal. In conclusion, the impaired IGFBP-1 response to meal-related hyperinsulinaemia in obese patients with T2DM suggests a decreased availability of active IGF-1, leading to a decrease in glucose uptake during and after a meal in these patients. The stimulated meal response to glucagon, which contributes to postprandial hyperglycaemia, could not explain the increase in serum IGFBP-1 in these obese patients with T2DM.
IGFs (insulin-like growth factors) induce both glucose and amino acid uptake [1–4]. The amount of circulating IGFs are abundant and the combined potential biological effects are 100-fold that of insulin ; however, IGF-1 circulates in plasma bound to IFGBPs [IGF-binding proteins; IGFBP-1–IGFBP-6] , which regulate the unbound biological forms. Unbound IGF-1 has a half-life of approx. 5 min . The binding to the IGFBPs ensures storage and a prolonged effect, as well as inhibition of the hypoglycaemic effect of IGF-1 [2,6]. The majority of IGF-1 is bound to IGFBP-3 which, together with an ALS (acid-labile subunit), forms a large ternary complex (150 kDa) that cannot leave the circulation, with a half life of >15 h [2,7]. Approx. 10–20% of IGF is bound to the smaller binding proteins IGFBP-1 and IGFBP-2, with a half-life in serum of 1–2 h . Less than 1% of the IGFs circulate in an unbound form, with a high affinity to one of these proteins IGFBP-1 . Moreover, IGFBP-1, although not the most predominant IGFBP, is the only binding protein to have a rapid regulation in vivo and appears to be a reservoir from which IGF-1 can be dissociated into unbound forms and which stabilizes circulating levels in serum [9,10]. IGFBP-1 is also a marker of unbound IGF-1 .
The regulation of circulating IGF-1 bioavailability is a key function of the IGFBPs . IGFBP-1 has been suggested to modulate the short-term bioavailability and activity of IGF-1 [9,10] and plays a role in glucose homoeostasis [12,13]. IGFBP-1 is secreted as a phosphoprotein , and the affinity of phosphorylated human IGFBP-1 for IGF-1 is 6-fold higher than for the non-phosphorylated protein . IGFBP-1 has been suggested to play a role in the acute regulation of IGF bioavailability by binding unbound IGFs, and the regulation of its affinity by phosphorylation may be of important and versatile metabolic function . Basal levels of IGFBP-1 can be low, normal or elevated in patients with T2DM (Type 2 diabetes mellitus) [16–18], whereas they are increased several-fold in patients with T1DM (Type 1 diabetes mellitus) [16,19]. IGFBP-1 is mainly produced by the liver in humans with a high production rate [19,20], and serum levels of IGFBP-1 have a diurnal rhythm , with the highest concentrations observed during the night and early morning; however, these are suppressed by food intake . Insulin regulates the production of IGFBP-1 by inhibiting its production at the transcriptional level both in vivo and in vitro [18,19,23,24]. Agents increasing intracellular levels of cAMP have been found to increase IGFBP-1 both at the transcriptional and post-transcriptional levels [25–27]. Glucagon, which mediates many of its cellular effects via cAMP, has been found to increase levels of circulating IGFBP-1 in vivo  and increase IGFBP-1 mRNA levels in vitro . During insulin infusion, IGFBP-1 is cleared from plasma with a half-life of 90–120 min in patients with T1DM and T2DM . It is not known how meal-induced hyperinsulinaemia affects the serum levels of IGFBP-1 or the bioavailability of IGF-1 in patients with T2DM. Recently, we have shown that the IGFBP-1 response to hyperinsulinaemia during an OGTT (oral glucose tolerance test) is decreased in obese patients with T2DM, as well as in patients with heredity T2DM, and that the decreased first-phase insulin response to an OGTT is associated with low levels in serum IGF-1 [30,31]. Postprandial hyperglycaemia could therefore be caused by hepatic insulin resistance also involving IGFBP-1 and indirectly bioactive IGF-1.
The aim of the present study was to evaluate the effect of a mixed meal on the serum levels of IGFBP-1 in obese patients with T2DM, non-obese patients with T1DM and healthy subjects, and to evaluate whether an elevated glucagon response to a mixed meal could explain the increase in serum IGFBP-1 in obese patients with T2DM despite hyperinsulinaemia. In order to achieve a normoglycaemic response to the meal in the patients with diabetes and to discriminate whether the results were related to hyperglycaemia, a Biostator® (artificial pancreas) was used.
MATERIAL AND METHODS
The study protocol was approved by the local Ethics Committee of Karolinska Hospital, and written informed consent was given by the subjects asked to participate in the study. Nine obese patients with T2DM and poor metabolic control, as reflected by their HbA1c (glycated haemoglobin) levels, and six non-obese T1DM patients participated, and their results were compared with that of nine healthy subjects. Gender, age, BMI (body mass index), duration of the disease and metabolic control of the participants are shown in Table 1. A renal function test was normal in all participants. No participants had signs of gastrointestinal disturbances or clinical evidence of neuropathy or proliferative retinopathy. NPH (neutral protamine hagedorn) insulin was interrupted 24 h before the experiment, and oral hypoglycaemic agents were withdrawn 3 weeks before the onset of the study. Blood glucose was controlled by the use of regular subcutaneously injected insulin up to 16 h before the experiments. All the subjects were studied after an overnight fast.
|.||Patients with .||.|
|Characteristic .||T1DM .||T2DM .||Healthy controls .|
|Gender (n) (male/female)||6/0||5/4||4/5|
|.||Patients with .||.|
|Characteristic .||T1DM .||T2DM .||Healthy controls .|
|Gender (n) (male/female)||6/0||5/4||4/5|
At 07.30 hours on the study day, three cannulae were inserted: one cannula placed retrograde was connected to a Biostator® (artificial pancreas) for continuous blood withdrawal to monitor blood glucose and for delivering insulin, a second cannula was inserted in a antecubital vein for intermittent blood sampling for analysis of hormone levels and was flushed with saline after use, and the third cannula was inserted in a contra lateral antecubital vein and was used for all of the infusions. At approx. 08.00 hours, the patients with diabetes were connected to the Biostator® to normalize blood glucose levels. The standard Biostator® controller algorithm was employed during feedback insulin infusion. The algorithms used in the Biostator® have been described in detail previously . Insulin infusion was started immediately after the first blood sample had been obtained. The meal-induced glycaemic responses in the patients with diabetes were clamped to that of the healthy subjects, approx. 4–5 mmol/l under basal conditions and approx. 6–7 mmol/l postprandially. Normoglycaemia was then achieved within a period of 20–60 min, and a standard lunch containing 2301 kJ (550 kcal) of total energy (comprising 28% protein, 26% fat and 46% carbohydrates) was served at 0 min. After approx. 40 min, normoglycaemia was achieved. The healthy subjects were studied using the same protocol, but without the Biostator® feedback insulin infusion. In addition, the nine patients with T2DM were studied as out-patients after an overnight fast during inhibition of glucagon secretion by continuous infusion with somatostatin (Sandostatin®; Sandoz) at a constant rate of 6.7 ng·min−1·kg−1 of body weight from −30 min and throughout the study.
Blood samples for the determination of glucose, insulin, C-peptide, glucagon, GH (growth hormone), somatostatin and IGFBP-1 were taken before the Biostator® was connected and during normoglycaemia at −30, 0, +15, +30, +60, +90, +120, +150 and +180 min following the meal.
Blood samples were collected in plastic tubes containing EDTA (0.35 mol/l) and Trasylol (1000 kallikrein inhibitor units; Bayer) and immediately placed on ice. Samples were centrifuged at 4 °C and plasma was frozen at −20 °C. Blood glucose was measured using a glucose oxidase method . HbA1c was measured using an isoelectric method  and was DCCT (Diabetes Control and Complications Trial)-aligned. GH levels was determined by RIA . The intra- and inter-assay CVs (coefficients of variation) for GH were 4 and 10% respectively, and the detection limit was 0.2 μg/l. Glucagon was measured by RIA, based on the method of Falcoona and Unger , using the antibody 30K. For this analysis, the inter-assay CV was 5%. Insulin was analysed by RIA using antibodies raised in our laboratory . The intra- and inter-assay CVs were 9 and 12% respectively, and the detection limit was 9 μ-units/l. C-peptide was assayed using a commercial kit (Novo Research Institute, Bagsvaerd, Denmark). Plasma concentrations of IGFBP-1 were measured using an RIA as described previously  with rabbit antisera from animals injected with pure IGFBP-1 isolated from human amniotic fluid. A polyclonal antibody was used, measuring both phosphorylated and non-phosphorylated forms of IGFBP-1. The detection limit of the assay was 3 ng/ml, and the intra- and inter-assay CVs were 4 and 11% respectively. The cross-reaction with other IGFBPs is <0.5%. Somatostatin was extracted and concentrated from 2 ml of plasma and was analysed using an RIA developed in our laboratory . The detection limit was 0.5 pg/assay tube, and the intra- and inter-assay CVs were 13 and 6% respectively. All blood samples from each subject were analysed in duplicate in the same assay run.
Statistical analysis and calculations
Results are means±S.E.M. Statistical significance was set at P<0.05 and was assessed using ANOVA with repeated measures for interaction with time and treatment (somatostatin compared with saline in the T2DM group). For paired data, a paired Student's t test was carried out. For comparisons between groups of patients, one-way ANOVA was applied or ANOVA on rank-transformed data for non-normally distributed variables. The meal-related glucose and hormonal responses were calculated as the integrated increments above the basal value starting 30 min before and up to 180 min after the meal. Insulin requirements for normalization of blood glucose were calculated from Biostator® readings. For the purpose of the calculation, undetectable GH and somatostatin levels were assigned values of 0.2 and 2.5 μg/l respectively.
Table 2 shows the basal levels of glucose, insulin, C-peptide, glucagon, GH, somatostatin and IGFBP-1. Basal fasting glucose levels were similar in patients with T1DM and obese patients with T2DM, whereas insulin levels were 3-fold higher in patients with T2DM compared with patients with T1DM (P<0.01). The basal fasting IGFBP-1 levels were increased more than 5-fold in the patients with T1DM than in the obese patients with T2DM (P<0.001) and healthy controls (26±10 μg/l; P<0.001). Basal C-peptide levels were significantly lower (P<0.001) in the patients with T1DM compared with the obese patients with T2DM. Glucagon levels were not significantly different in the two groups of patients with diabetes, although there was a tendency for higher glucagon levels in the patients with T1DM. Neither somatostatin nor GH levels differed significantly between the two groups, although higher levels of GH were seen in some of the patients with T1DM (Table 2).
|.||Patients with .|
|Characteristic .||T1DM .||T2DM .|
|.||Patients with .|
|Characteristic .||T1DM .||T2DM .|
Effect of insulin infusion before the meal
The glycaemic, hormonal and binding-protein responses to the first phase of insulin infusion (−30 min) are shown in Figure 1. Blood glucose decreased significantly from basal levels in patients with T2DM (from 13.7±1.6 to 4.6±0.2 mmol/l; P<0.001) and T1DM (from 15.2±2.5 to 4.6±0.2 mmol/l; P<0.01). Circulating insulin levels increased from basal levels in patients with T2DM (from 30.9±4 to 68.7±17 m-units/l; P=not significant) and T1DM (from 10.2±1 to 28.2±5 m-units/l; P<0.01). C-peptide levels were decreased by 50% in patients with T2DM (from 0.87±0.08 to 0.42±0.04 nmol/l; P<0.0001). IGFBP-1 decreased significantly by 26% in patients with T1DM (from 184±2 to 135±17 μg/l; P<0.05) and by 41% in patients with T2DM (from 36±7 to 21.2±3.2 μg/l; P<0.05). Glucagon (Figure 1D), somatostatin (results not shown) and GH (results not shown) concentrations did not change significantly in either patients with T1DM or T2DM
Serum concentrations of glucose (A), insulin (B), IGFBP-1 (C) and glucagon (D) before and during a standard meal in healthy controls (▲), non-obese patients with T1DM (○) and obese patients with T2DM (□)
Before the meal at 0 min, the mean levels of glucagon (Figure 1D) and GH (results not shown) were not significantly different between the groups. The glucose (Figure 1A), IGFBP-1 (Figure 1C) and C-peptide (results not shown) levels were similar in the obese patients with T2DM and healthy controls. IGFBP-1 was increased more than 5-fold in patients with T1DM compared with obese patients with T2DM and healthy controls (Figure 1C). Insulin levels were significantly increased in the patients with T2DM (49.3±11.6 m-units/l) compared with the healthy controls (11.2±1.4 m-units/l; P<0.001) and patients with T1DM (28.2±5 m-units/l; P<0.01).
Response to a standard meal
The peak glucose response (approx. 6.5 mmol/l in all groups) to the meal was seen 30 min after the start of the meal (Figure 1A). The peak insulin levels, observed at 30–60 min, were similar in patients with T1DM (77±29 m-units/l) and healthy controls (55±3 m-units/l), whereas they were more than 3-fold higher in patients with T2DM (196±59 m-units/l) than in the controls with a delay of approx. 30 min (Figure 1B). The C-peptide levels increased 2-fold in the controls, but not significantly in the patients with T2DM (results not shown). IGFBP-1 decreased in the patients with T1DM and healthy controls with a similar clearance rate, the half-life being approx. 120 min. No significant decrease in IGFBP-1 was observed in the patients with T2DM (Figures 1C and 1Ci). Glucagon increased significantly during the first 30 min in both patients with T1DM and T2DM and within 90 min in the healthy controls, with sustained, but not significantly higher, levels in patients with T2DM (Figure 1D). The somatostatin levels increased significantly in all of the study groups in response to the meal, with the highest levels being in the healthy controls (results not shown). GH levels decreased in all subjects with a nadir at 60 min, whereafter a significant increase was observed only in the healthy controls at the end of the study (results not shown).
Response to somatostatin infusion
During 210 min of somatostatin infusion in the nine obese patients with T2DM, somatostatin increased to approx. 100 pg/ml (Figure 2A), and the glucagon response to the meal was suppressed by approx. 150% (Figure 2B). Glucose levels were significantly (P<0.01) lower at +15 and +30 min during somatostatin infusion (Figure 2D). Insulin levels with somatostatin infusion were significantly lower at +30 min compared with the saline control (126±14 and 63±6 m-units/l respectively; P<0.05), after which no differences were observed (Figure 2C). C-peptide levels were significantly lower from +15 to +180 min (results not shown). IGFBP-1 levels increased concomitantly with the peak in insulin, without any significant decline (Figure 2E). Moreover, the peaks in both glucose and IGFBP-1 were shifted to the right. Somatostatin infusion during a mixed meal induced a significant increase at 90–180 min in IGFBP-1 (P<0.05), despite a several-fold increase in insulin levels and inhibited glucagon secretion.
Serum concentrations of somatostatin (A), glucagon (B), insulin (C), glucose (D) and IGFBP-1 (E) before and during a standard meal in patients with T2DM without (□) or with (■) an infusion of somatostatin
The present study confirms the meal-related insulin resistance in obese patients with T2DM [39–41] and shows that these patients respond differently to meal-related hyperinsulinaemia with an elevated glucagon response compared with patients with T1DM and healthy controls [28,32,42], although having a normoglycaemic response during meal. It has been suggested that the inappropriate excessive secretion of glucagon in patients with T2DM is mediated because of α-cell insulin resistance in T2DM . These results support our findings in patients with T2DM showing a high postprandial glucagon level, despite a several-fold increase in insulin during the meal. However, the patients with T1DM also had a high glucagon response despite hyperinsulinaemia during meal, which could be explained by an insufficient insulin concentration locally in the islets. Moreover, basal fasting glucagon levels were higher in patients with T1DM compared with T2DM, which may be due to insulin deficiency. A new finding in the obese patients with T2DM is the impaired meal-related response of IGFBP-1, with no suppression despite a several-fold increase in circulating insulin. We [12,16] and others [15,18,44] have shown that fasting IGFBP-1 is inversely correlated with insulin concentrations in serum from healthy subjects and patients with T1DM; however, in obese patients with T2DM, there is no such correlation [44,45]. Patients with T2DM have basal insulin production, but a decreased insulin response to glucose and meal-induced insulin resistance [39,46]. The cause of this is not known, but an impaired first phase insulin response  and an abnormal glucagon response to a meal may be one explanation [39,46]. Postprandial hyperglycaemia in patients with T2DM is an early event in the disease and is responsible for increased cardiovascular disease morbidity and mortality . Our present findings suggest that obese patients with T2DM have a marked hepatic insulin resistance during a meal, encompassing both the effect of insulin on gluconeogenesis and the glucose counter-regulatory hormone glucagon  and on IGFBP-1 . These findings also indirectly suggest that postprandial hyperglycaemia in obese patients with T2DM is the effect of decreased bioavailable IGF-1 as well as hepatic insulin resistance during the meal. Interestingly, the IGFBP-1 response to insulin before the meal was adequate.
Fasting levels of insulin were 180% higher in obese patients with T2DM compared with the healthy controls; however, fasting IGFBP-1 levels was not significantly lowered. In healthy individuals and patients with T1DM, circulating IGFBP-1 levels had a characteristic diurnal pattern, with mealtime suppression related to increases in insulin concentrations and gradual increases during fasting [50–52]. This diurnal rhythm is absent in patients with Cushing's disease . Thus the inverse regression between fasting insulin and IGFBP-1 shown previously [21,44,54] was shifted to the right, suggesting relative hepatic insulin resistance or different or unknown factors in patients with T2DM that stimulate the production and/or degradation of IGFBP-1. The relative postprandial increase in IGFBP-1 and glucose, despite high insulin levels, may also suggest hepatic insulin resistance in obese patients with T2DM . The elevated glucagon levels observed in obese patients with T2DM during the meal could have contributed to the augmented IGFBP-1 levels. Studies have shown that glucagon stimulates the release of IGFBP-1 in vivo  and in fetal liver cells in vitro ; however, suppression of glucagon release with somatostatin (Sandostatin®) in our present study did not blunt the increase in IGFBP-1. In fact, somatostatin infusion increased IGFBP-1 levels further compared with healthy controls, despite high circulating insulin levels and a decreased level of glucagon after a 15 min delay. The 15 min delay in the glucose peak observed in our present study could be secondary to an altered gastric motility by somatostatin infusion, and may also explain the delayed response of IGFBP-1 during the somatostatin infusion. However, it would not explain the paradoxical postprandial increase in IGFBP-1. In a previous study , we have shown that hyperinsulinaemia in healthy subjects and in patients with T1DM caused a decrease in IGFBP-1, despite low C-peptide levels. We cannot exclude, however, that somatostatin may have a direct effect on IGFBP-1  and neither can we exclude that acute hyperinsulinaemia causes a transient impaired hepatic insulin extraction . It is also possible that other unidentified factor(s), possibly gastrointestinal peptide(s), released during the meal in patients with T2DM may be able to stimulate the hepatic production of IGFBP-1. Theoretical alternative explanations are that there may be an activated liver cytokine pathway involved in patients with insulin resistance and obesity , which may contribute to a direct stimulation of IGFBP-1 , although this is still unproven. It has been shown that increased IGFBP-1 concentrations in the circulation decrease the bioavailability of IGF-1, inhibiting the hypoglycaemic effect of IGF-1 . This hypothesis is supported by the present findings, in obese patients with T2DM, of sustained increased glucose levels for 3 h after the meal, despite high insulin levels after the increase in IGFBP-1 (Figure 2). IGF-1 bioavailability was also low in patients with GH deficiency  and untreated thyrotoxicosis with signs of insulin resistance, but increased markedly after replacement therapy as pharmacotherapy induces euthyroidism. The glucose level in that same study  was significantly higher during hyperthyroidism with low IGF-1 bioavailability, but was normalized when patients became euthyroidic . In vivo studies in rats with rIGFBP-1 (recombinant IGFBP-1) have shown that it causes an increase in blood glucose and blocks the effect of IGF-1 .
It has also been shown that treatment with IGF-1 may decrease insulin resistance in patients with T2DM and lowers blood glucose while, at the same time, lowering serum insulin levels in healthy volunteers. This mechanism appears to be independent of the activation of the insulin receptor . Small-scale clinical trials have demonstrated the potential use of rhIGF-1 (recombinant human IGF-1) in selected cases of severe insulin resistance . It is not excluded that the relative lowering of IGF-1 bioavailability observed in the present study may contribute to meal-related insulin resistance in obese patients with T2DM.
In summary, our present study verifies that obese patients with T2DM have an impaired meal-related suppression of IGFBP-1, despite a several-fold increase in circulating insulin, and this correlation does not appear to be dependent on the meal-related glucagon level, suggesting postprandial hepatic insulin resistance in obese patients with T2DM, which may explain the postprandial increase in glucose.
We dedicate this paper to the memory of Mark K. Gutniac MD, PhD, a great inspiration and friend. We thank Berit Rylander for excellent technical assistance. This work was supported by Grants from the Swedish Medical Research Council (Grant No.4224), The Swedish Diabetes Association and the Family Erling-Persson Foundation and Tore and Ragnar Söderbergs Foundation.