Michael P. Brugts

Low Circulating Insulin-Like Growth Factor I Bioactivity in Elderly Men Is Associated with Increased Mortality

CONTEXT Low IGF-I signaling activity prolongs lifespan in certain animal models, but the precise role of IGF-I in human survival remains controversial. The IGF-I kinase receptor activation assay is a novel method for measuring IGF-I bioactivity in human serum. We speculated that determination of circulating IGF-I bioactivity is more informative than levels of immunoreactive IGF-I. OBJECTIVE Our objective was to study IGF-I bioactivity in relation to human survival. DESIGN, SETTING, AND STUDY PARTICIPANTS: We conducted a prospective observational study at a clinical research center at a university hospital of 376 healthy elderly men (aged 73-94 yr). MAIN OUTCOME MEASURES IGF-I bioactivity was determined by the IGF-I kinase receptor activation assay. Total and free IGF-I were determined by IGF-I immunoassays. Mortality was registered during follow-up (mean 82 months). RESULTS During the follow-up period of 8.6 yr, 170 men (45%) died. Survival of subjects in the highest quartile of IGF-I bioactivity was significantly better than in the lowest quartile, both in the total study group [hazard ratio (HR) = 1.8; 95% confidence interval (95% CI) = 1.2-2.8; P = 0.01] as well as in subgroups having a medical history of cardiovascular disease (HR = 2.4; 95% CI = 1.3-4.3; P = 0.003) or a high inflammatory risk profile (HR = 2.3; 95% CI = 1.2-4.5; P = 0.01). Significant relationships were not observed for total or free IGF-I. CONCLUSION Our study suggests that a relatively high circulating IGF-I bioactivity in elderly men is associated with extended survival and with reduced cardiovascular risk.

Normal Values of Circulating Insulin-Like Growth Factor-I Bioactivity in the Healthy Population: Comparison with Five Widely Used IGF-I Immunoassays

BACKGROUND IGF-I immunoassays are primarily used to estimate IGF-I bioactivity. Recently an IGF-I-specific kinase receptor activation assay (KIRA) has been developed as an alternative method. However, no normative values have been established for the IGF-I KIRA. OBJECTIVE The objective of the study was to establish normative values for the IGF-I KIRA in healthy adults. DESIGN This was a cross-sectional study in healthy nonfasting blood donors. STUDY PARTICIPANTS Participants included 426 healthy individuals (310 males, 116 females; age range 18-79 yr). MAIN OUTCOME MEASURES IGF-I bioactivity determined by the KIRA was measured. Results were compared with total IGF-I, measured by five different IGF-I immunoassays. RESULTS Mean (+/- sd) IGF-I bioactivity was 423 (+/- 131) pmol/liter and decreased with age (beta = -3.4 pmol/liter.yr, P < 0.001). In subjects younger than 55 yr, mean IGF-I bioactivity was significantly higher in women than men. Above this age this relationship was inverse, suggesting a drop in IGF-I bioactivity after menopause. This drop was not reflected in total IGF-I levels. IGF-I bioactivity was significantly related to total IGF-I (r(s) varied between 0.46 and 0.52; P < 0.001). CONCLUSIONS We established age-specific normative values for the IGF-I KIRA. We observed a significant drop in IGF-I bioactivity in women between 50 and 60 yr, which was not perceived by IGF-I immunoassays. The IGF-I KIRA, when compared with IGF-I immunoassays, theoretically has the advantage that it measures net effects of IGF-binding proteins on IGF-I receptor activation. However, it has to be proven whether information obtained by the IGF-I KIRA is clinically more relevant than measurements obtained by IGF-I immunoassays.

IGF-I Bioactivity in an Elderly Population: Relation to Insulin Sensitivity, Insulin Levels, and the Metabolic Syndrome

OBJECTIVE There is a complex relationship between IGF-I, IGF binding proteins, growth hormone, and insulin. The IGF-I kinase receptor activation assay (KIRA) is a novel method for measuring IGF-I bioactivity in human serum. We speculated that determination of IGF-I bioactivity might broaden our understanding of the IGF-I system in subjects with the metabolic syndrome. The purpose of our study was to investigate whether IGF-I bioactivity was related to insulin sensitivity and the metabolic syndrome. RESEARCH DESIGN AND METHODS We conducted a cross-sectional study embedded in a random sample (1,036 elderly subjects) of a prospective population-based cohort study. IGF-I bioactivity was determined by the IGF-I KIRA. Categories of glucose (in)tolerance were defined by the 2003 American Diabetes Association criteria. Insulin sensitivity was assessed by homeostasis model assessment. The Adult Treatment Panel III definition of the metabolic syndrome was used. RESULTS In subjects with normal fasting glucose and impaired fasting glucose, IGF-I bioactivity progressively increased with increasing insulin resistance, peaked at fasting glucose levels just below 7.0 mmol/l, and dropped at higher glucose levels. Mean IGF-I bioactivity peaked when three criteria of the metabolic syndrome were present and then declined significantly when five criteria of the metabolic syndrome were present. CONCLUSIONS We observed that IGF-I bioactivity was related to insulin sensitivity, insulin levels, and the metabolic syndrome. Our study suggests that there exists an inverse U-shaped relationship between IGF-I bioactivity and number of components of the metabolic syndrome. This observation contrasts with previous results reporting an inverse relationship between total IGF-I and components of the metabolic syndrome.

Insulin glargine is more potent in activating the human IGF-I receptor than human insulin and insulin detemir

OBJECTIVE To investigate whether human insulin (HI) and insulin analogues differ in their ability to activate the human IGF-I receptor (IGF-IR), the human insulin receptor A (IR-A) and the human insulin receptor B (IR-B) in vitro. METHODS HI, short-acting insulin analogues (insulin aspart; insulin lispro) and long-acting insulin analogues (insulin glargine; insulin detemir) were compared by using kinase receptor activation (KIRA) bioassays specific for IGF-IR, IR-A or IR-B, respectively. These assays quantify ligand activity by measuring receptor auto-phosphorylation upon ligand binding. HI and insulin analogues were tested in a range from 0.1 to 100 nM. RESULTS Short-acting analogues: Overall, short-acting insulin analogues did not differ substantially from HI, nor from each other. Insulin lispro was slightly more potent than HI and insulin aspart in activating the IGF-IR, only reaching statistical significance at 100 nM (p<0.01). Long-acting analogues: At <10 nM insulin glargine was as potent as HI in activating the IRs and IGF-IR. At 10-100 nM insulin glargine was significantly more potent than HI in activating the IR-B (p<0.05) and IGF-IR (p<0.001). Insulin glargine was more potent than insulin detemir in activating all three receptors (p<0.001). Insulin detemir was less potent than HI in activating the IRs at 1-10 nM (p<0.01) and IGF-IR at >1 nM (p<0.05). CONCLUSIONS Insulin glargine was more potent in activating the IGF-IR than HI and insulin detemir. Since KIRA bioassays do not mimic the exact in vivo situation, further research is needed to find out whether our data have implications for clinical use of insulin glargine.

Effect of intensive insulin therapy on the somatotropic axis of critically ill children

CONTEXT Intensive insulin therapy (IIT) improved outcome in the adult and pediatric intensive care unit (PICU) compared with conventional insulin therapy (CIT). IIT did not increase the anabolic hormone IGF-I in critically ill adults, but feeding in critically ill children and pediatric hormonal responses may differ. Twenty-five percent of the children with IIT experienced hypoglycemia, which may have evoked counterregulatory responses. OBJECTIVE We hypothesized that IIT reactivates the somatotropic axis and anabolism in PICU patients. DESIGN This was a preplanned subanalysis of a randomized controlled trial on IIT. PATIENTS We studied 369 patients who stayed in PICU for at least 3 d (study 1) and 126 patients in a nested case-control study (study 2). MAIN OUTCOME MEASURES Circulating insulin, C-peptide, GH, IGF-I, bioavailable IGF-I, IGF-binding protein (IGFBP)-1, IGFBP-3, and acid-labile subunit were analyzed upon admission and d 3. In the nested case-control study, the somatotropic axis, cortisol, and glucagon were analyzed before and after hypoglycemia. RESULTS On d 3, C-peptide was more than 10-fold lower (P < 0.0001) in the IIT group than in the CIT group. IIT increased circulating GH (P = 0.04) and lowered bioavailable IGF-I (P = 0.002). IIT also decreased IGFBP-3 (P = 0.0005) and acid-labile subunit (P = 0.007), while increasing IGFBP-1 (P = 0.04) and the urea/creatinine ratio, a marker of catabolism (P = 0.03). In the nested case-control study, IGFBP-1 was increased after hypoglycemia, whereas the somatotropic axis and the counterregulatory hormones cortisol and glucagon did not change. CONCLUSIONS Despite improved PICU outcome, IIT did not counteract the catabolic state of critical illness. Suppression of portal insulin may have resulted in lower bioavailable IGF-I.

Insulin-Like Growth Factor-I Receptor Stimulating Activity is Associated with Dementia

BACKGROUND Insulin-like growth factor-I (IGF-I) is a pleiotropic hormone. Several studies have related IGF-I levels to dementia, but evidence remains inconclusive. IGF-I receptor stimulating activity is a more direct measure of biologically available IGF-I than total IGF-I levels. OBJECTIVE To investigate whether IGF-I receptor stimulating activity is associated with prevalent and incident dementia. METHODS IGF-I receptor stimulating activity was measured using an IGF-I kinase receptor activation assay in 1,014 persons from the Rotterdam Study. Dementia was assessed at baseline (1997-1999) and continuously during follow-up until September 2011. Associations of IGF-I receptor stimulating activity with prevalent dementia were investigated using logistic regression and with incident dementia using Cox proportional hazards models. All models were adjusted for age and gender, and additionally for hypertension, glucose, waist circumference, APOE-ε4 carrier status, total cholesterol, and HDL-cholesterol. RESULTS Thirty participants had prevalent dementia and during 8,589 person-years of follow-up, 135 persons developed incident dementia. A higher level of IGF-I receptor stimulating activity was associated with a higher prevalence of dementia (fully adjusted odds ratio 1.47; 95% CI 1.10-1.97) and with a higher risk of incident dementia (fully adjusted hazard ratio 1.15; 95% CI 1.00-1.33). Similar associations were found for Alzheimers disease and in persons without diabetes mellitus. CONCLUSIONS Higher levels of IGF-I receptor stimulating activity are associated with a higher prevalence and with a higher incidence of dementia. These results suggest that IGF-I increases in response to neuropathological changes in dementia and could reflect a state of IGF-I resistance in dementia.

Heterophilic antibodies may be a cause of falsely low total IGF1 levels

BACKGROUND A low serum total IGF1 is considered as a diagnostic indicator of GH deficiency (GHD) in the presence of hypopituitarism. Introduction of IRMA and chemiluminescent immunometric assay (CLIA) IGF1 immunoassays has introduced endogenous antibodies as a new source of interference. In general, this goes unnoticed and might lead to unnecessary diagnostic and therapeutic interventions. CASE A 56-year-old man was referred with a decline in physical performance, unexplained osteopenia, and weight loss of 3 kg over the past 8 months. Although clinical signs and symptoms were unremarkable, laboratory results pointed to secondary hypothyroidism and secondary hypogonadism. In addition, the serum total IGF1 level (CLIA; Siemens Medical Solutions Diagnostics) was in the low normal range. Two GH stimulation tests were performed, but these tests did not support the diagnosis GHD. Moreover, IGF1 bioactivity measured by the kinase receptor activation assay was normal. Interference of heterophilic antibodies was considered. After pretreatment with specific heterophilic blocking tubes that contain blocking reagents to eliminate heterophilic antibodies, serum-free thyroxine, testosterone, and IGF1 levels turned out to be normal. CONCLUSION To the best of our knowledge, we here describe the first case in the literature of a patient with low serum total IGF1 levels due to interference from heterophilic antibodies in the used IGF1 immunoassay. When confronted with low-IGF1 levels that do not fit the clinical picture, interference of heterophilic antibodies should be considered in the differential diagnosis.

Circulating insulin-like growth factors may contribute substantially to insulin receptor isoform A and insulin receptor isoform B signalling.

BACKGROUND Only a fraction of circulating insulin-like activity is due to insulin itself. The aim of this study was to determine total serum insulin-like activity mediated via the insulin receptor isoform A (IR-A) and isoform B (IR-B) by using kinase receptor activation (KIRA) assays specific for the IR-A and IR-B. METHODS The IR-A and IR-B KIRA assays use human embryonic kidney cells which have been transfected with the human IR-A or IR-B gene and quantify serum-mediated phosphorylation of the IR. RESULTS Both IR KIRA assays were sensitive (detection limit 32 pmol/L) and precise (intra- and inter assay CV: <12% and <15%). The EC₅₀s of insulin, IGF-I and IGF-II were 1.4, 11.2 and 6.7 nmol/L for the IR-A KIRA assay, and 1.3, 31.0 and 15.7 nmol/L for the IR-B KIRA assay. The operational range of both assays allowed for determination of total insulin-like activity in human serum. Analysis of serum samples showed that there was a significant positive correlation between serum insulin-like and immunoreactive insulin concentrations (IR-A: r = 0.56, p = 0.01, IR-B: r = 0.68, p = 0.001). Importantly, addition of IGF-I or IGF-II antibodies to human serum samples could substantially decrease the endpoint signal in both KIRA assays. CONCLUSIONS We showed that serum IGF-I and IGF-II may substantially contribute to IR signalling. Since IR isoform specific KIRA assays also take into account the contribution of IGFs present in serum on IR signalling, they may help to gain more insight into the roles of IGF mediated IR-A and IR-B activation in health and disease.

High IGFBP2 levels are not only associated with a better metabolic risk profile but also with increased mortality in elderly men

OBJECTIVE Serum IGF-binding protein 2 (IGFBP2) concentrations are reduced in obese humans and increase after a prolonged period of fasting. We investigated the association between IGFBP2 levels and mortality together with other factors that are related to IGFBP2, including the metabolic syndrome and physical function. DESIGN A prospective observational study at a clinical research center of 403 independently living elderly men (aged 73-94 years). METHODS Mortality was registered during 8.6 years of follow-up. Physical performance score (PPS), grip strength (GS), and bone mineral density (BMD) were measured. The measurements taken a baseline were: IGF1; IGFBP1, -2, and -3; IGF1 bioactivity; triiodothyronine (T(3)); and reverse T(3). Further, BMI, insulin sensitivity, cholesterol, inflammatory markers, and albumin levels were also measured. RESULTS During the follow-up, 180 men died. Higher PPS, GS, and BMD were independently related to a reduced mortality (hazard ratio (HR)=0.87/point, 95% confidence interval (95% CI)=0.82-0.91, P<0.001; HR=0.96/kp, 95% CI 0.94-0.98, P<0.001; and HR=0.21/(g/cm(2)), 95% CI 0.07-0.61, P<0.01). Higher serum IGFBP2 levels were strongly related to mortality (HR=2.26/(mg/l), 95% CI 1.57-3.27, P<0.001). This was independent of comorbidity, physical function, IGF1 bioactivity, and other somatotropic parameters, including BMI and the metabolic syndrome. In addition, IGFBP2 levels were higher in subjects with nonthyroidal illness, and higher IGFBP2 levels were significantly associated with lower albumin concentrations. CONCLUSION Despite the strong relationship between high IGFBP2 and low physical function, both were strongly and independently related to increased 8-year mortality in elderly men. IGFBP2 may be a useful biomarker integrating the nutritional status, as well as the biological effects of GH, IGF1, and insulin.

Somatostatin Analogues for Receptor Targeted Photodynamic Therapy

Photodynamic therapy (PDT) is an established treatment modality, used mainly for anticancer therapy that relies on the interaction of photosensitizer, light and oxygen. For the treatment of pathologies in certain anatomical sites, improved targeting of the photosensitizer is necessary to prevent damage to healthy tissue. We report on a novel dual approach of targeted PDT (vascular and cellular targeting) utilizing the expression of neuropeptide somatostatin receptor (sst2) on tumor and neovascular-endothelial cells. We synthesized two conjugates containing the somatostatin analogue [Tyr3]-octreotate and Chlorin e6 (Ce6): Ce6-K3-[Tyr3]-octreotate (1) and Ce6-[Tyr3]-octreotate-K3-[Tyr3]-octreotate (2). Investigation of the uptake and photodynamic activity of conjugates in-vitro in human erythroleukemic K562 cells showed that conjugation of [Tyr3]-octreotate with Ce6 in conjugate 1 enhances uptake (by a factor 2) in cells over-expressing sst2 compared to wild-type cells. Co-treatment with excess free Octreotide abrogated the phototoxicity of conjugate 1 indicative of a specific sst2-mediated effect. In contrast conjugate 2 showed no receptor-mediated effect due to its high hydrophobicity. When compared with un-conjugated Ce6, the PDT activity of conjugate 1 was lower. However, it showed higher photostability which may compensate for its lower phototoxicity. Intra-vital fluorescence pharmacokinetic studies of conjugate 1 in rat skin-fold observation chambers transplanted with sst2 + AR42J acinar pancreas tumors showed significantly different uptake profiles compared to free Ce6. Co-treatment with free Octreotide significantly reduced conjugate uptake in tumor tissue (by a factor 4) as well as in the chamber neo-vasculature. These results show that conjugate 1 might have potential as an in-vivo sst2 targeting photosensitizer conjugate.