Rikke Hjortebjerg

Determination of IGFs and their binding proteins.

The worldwide clinical and scientific interest in peptides belonging to the insulin-like growth factor (IGF) system has brought along a call for standardization of assays used to quantify the different IGF related proteins. This relates in particular to the measurement of IGF-I, which has stood the test of time as an important biochemical tool in the diagnosis and treatment of growth hormone (GH) related disorders. The first international consensus statement on the measurement of IGF-I in 2011 represents an important milestone and will undoubtedly improve commutability of reference ranges for IGF-I and clinically applicable cut-off values. By contrast, there is no consensus addressing the measurements of the other IGF-related peptides. Nevertheless, measurement of these peptides may be of interest, either as additional tools in GH disorders or as prognostic biomarkers of various diseases. Therefore, standardization of assays for the other IGF-related peptides is highly relevant. This chapter discusses the recent consensus on IGF-I measurements and how this approach may be applied to measurement of the other IGF-related peptides. In addition, assay pitfalls, pre- and post-analytical challenges, alternative methods for IGF-I measurements and potential assays of tomorrow will be discussed.

Insulin growth factor binding proteins as therapeutic targets in type 2 diabetes.

Introduction: The signaling pathways of the insulin-like growth factors (IGFs) have been implicated in the aetiology of type 2 diabetes (T2D) and a number of therapeutic modalities aiming at the IGF-axis have been considered. Administration of IGF-I has been reported to improve insulin sensitivity in healthy subjects and patients with T2D. In recent years, the IGF binding proteins (IGFBPs) have also been associated with metabolic disorders, prompting the idea that IGFBPs play important roles in the pathogenesis of T2D. Thus, by virtue of their role in the regulation of IGF effects, the IGFBPs have emerged as potential biomarkers and therapeutic targets in metabolic syndromes and T2D. Areas covered: The article provides an overview on recent findings in clinical and experimental IGFBP-research and addresses the studies that have investigated the potentials of the IGFBPs as therapeutic targets in T2D. Expert opinion: There is plenty of therapeutic promise within the IGF system, but further understanding of the IGFs in T2D is necessary to avoid off-target effects. Strong evidence supports the use of IGFBPs as therapeutic targets in the treatment of T2D, and it is not difficult to foresee the use of IGFBPs as part of a combination therapy alongside other anti-diabetic drugs.

PAPP-A and IGFBP-4 fragment levels in patients with ST-elevation myocardial infarction treated with heparin and PCI.

OBJECTIVES Circulating levels of pregnancy-associated plasma protein-A (PAPP-A) predict outcome in patients with acute coronary syndrome (ACS). Unfortunately, administration of heparin to patients with ACS increases circulating PAPP-A, probably by a detachment of PAPP-A from cell surfaces, inducing a considerable bias when using PAPP-A as a biomarker. It remains unknown whether PAPP-A-derived N- and C-terminal fragments of insulin-like growth factor binding protein-4 (NT-IGFBP-4/CT-IGFBP-4) are acutely affected by the increase in PAPP-A. METHODS We prospectively included 78 patients with ST-segment elevation myocardial infarction (STEMI) treated with percutaneous coronary intervention (PCI). Prior to PCI, patients were injected with 10,000IU of unfractionated heparin (UFH). Blood samples were collected immediately before PCI, but after UFH-injection, immediately after PCI and on day 1 and day 2. Plasma IGFBP-4, CT-IGFBP-4 and NT-IGFBP-4 levels were determined by specific, novel immunoassays, and PAPP-A and IGF-I by commercial immunoassays. RESULTS Plasma PAPP-A was strongly elevated upon STEMI, UFH-administration and PCI with mean concentrations (95%-confidence interval) pre-PCI, post-PCI, day 1, and day 2 of 13.0 (11.2;15.2), 14.8 (13.1;16.8), 1.03 (0.90;1.18), and 1.08 (0.92;1.28) μg/L, respectively (p<0.0001). Pre-PCI concentrations of IGFBP-4, CT-IGFBP-4 and NT-IGFBP-4 were 154 (142;166), 53 (47;60) and 136 (122;150) μg/L, and levels were unaltered post-PCI. Concentrations increased on day 1 by 63 (43;87)%, 69 (36;110)%, and 47 (21;79)%, respectively (p<0.0001), i.e. at a time point when PAPP-A levels had normalized. CONCLUSION Plasma IGFBP-4-fragment levels are not acutely altered in patients with STEMI treated with UFH and PCI. Thus, they possess potentials as prognostic markers in ACS patients.

IGFBP-4 Fragments as Markers of Cardiovascular Mortality in Type 1 Diabetes Patients With and Without Nephropathy

CONTEXT Type 1 diabetes (T1D) is characterized by an increased risk of macrovascular complications. Pregnancy-associated plasma protein-A (PAPP-A) generated N- and C-terminal fragments of IGF binding protein-4 (NT-IGFBP-4 and CT-IGFBP-4) have been suggested as cardiac biomarkers. OBJECTIVE The objective of the study was to investigate the prognostic value of IGFBP-4 fragments in a cohort of T1D patients. DESIGN AND PATIENTS We prospectively followed up 178 T1D patients with diabetic nephropathy and 152 T1D patients with normoalbuminuria for 12.6 (range 0.2-12.9) years. MAIN OUTCOME MEASURES Levels of IGF-1, IGF-2, IGFBP-1-4, NT- and CT-IGFBP-4, and PAPP-A at baseline. RESULTS During follow-up, 15 patients with normoalbuminuria and 71 patients with nephropathy died. Of these deaths, 8 and 45 were due to fatal cardiovascular events, respectively. Using receiver-operating characteristic curve analyses, patients were divided into subgroups using cutoff values of 261 μg/L NT-IGFBP-4, 81 μg/L CT-IGFBP-4, or 10 mIU/L PAPP-A. All-cause mortality was significantly higher in patients with NT-IGFBP-4 levels (55% vs 16%, P < .001) and CT-IGFBP-4 levels (44% vs 15%, P < .001) above vs below cutoffs. Similarly, cardiovascular mortality was elevated in patients with high NT-IGFBP-4 levels (40% vs 7.8%, P < .001) and high CT-IGFBP-4 levels (30% vs 7.4%, P < .001). After adjustments for nephropathy and traditional cardiovascular risk factors, high NT- and CT-IGFBP-4 levels remained prognostic of cardiovascular mortality with hazard ratios [95% confidence interval (CI)] of 5.81 (95% CI 2.62-12.86) (P < .001) and 2.58 (95% CI 1.10-6.10) (P = .030), respectively. After adjustments, PAPP-A was not associated with overall or cardiovascular death. All IGF protein levels were higher in patients with diabetic nephropathy (P < .001), but no variables associated with mortality. CONCLUSION High IGFBP-4 fragment levels were associated with increased all-cause and cardiovascular mortality rates in T1D patients with and without diabetic nephropathy.

Insulin‐Like Growth Factor Binding Protein 4 Fragments Provide Incremental Prognostic Information on Cardiovascular Events in Patients With ST‐Segment Elevation Myocardial Infarction

Background Fragments of insulin‐like growth factor binding protein 4 (IGFBP‐4) are potential new biomarkers for cardiac risk assessment. The fragments are generated on specific cleavage by pregnancy‐associated plasma protein‐A, which exerts proatherogenic activity. This study investigated the prognostic value of IGFBP‐4 fragments in patients with ST‐segment elevation myocardial infarction. Methods and Results We prospectively included 656 patients with ST‐segment elevation myocardial infarction treated with percutaneous coronary intervention from September 2006 to December 2008. Blood samples were drawn before percutaneous coronary intervention, and levels of intact IGFBP‐4 and N‐terminal and C‐terminal IGFBP‐4 fragments were measured by specific assays. End points were 5‐year all‐cause and cardiovascular mortality and the combined end point of major adverse cardiac events. Prognostic potential was evaluated on top of a clinical model in terms of discrimination, calibration, and reclassification analysis. During follow‐up, 166 patients experienced a major adverse cardiac event and 136 patients died, of whom 69 died from cardiovascular causes. Both IGFBP‐4 fragments were associated with all end points (P<0.001). After multivariable adjustments, both N‐terminal and C‐terminal IGFBP‐4 fragment levels remained associated with all end points, including cardiovascular mortality with hazard ratios per doubling in protein concentration of 2.54 (95% CI 1.59–4.07; P<0.001) and 2.07 (95% CI 1.41–3.04; P<0.001), respectively. Incorporation of IGFBP‐4 fragments into a clinical model with 15 risk factors improved C‐statistics and model calibration and provided incremental prognostic contribution, as assessed by net reclassification improvement and integrated discrimination improvement. Conclusions IGFBP‐4 fragments are associated with increased risk of all‐cause mortality, cardiovascular mortality, and major adverse cardiac events in patients with ST‐segment elevation myocardial infarction.

The IGF system in patients with type 2 diabetes: associations with markers of cardiovascular target organ damage

OBJECTIVE Perturbations in the insulin-like growth factor (IGF) system may contribute to the accelerated cardiovascular disease (CVD) that occurs in patients with type 2 diabetes (T2D). However, it remains unknown whether the IGF system is also involved in the development of early, subclinical CVD. We characterised the IGF system in T2D patients and matched controls and examined the associations with markers of subclinical target organ damage. METHODS The study included 99 patients with recently diagnosed T2D and 99 age- and sex-matched controls. IGF-1 and IGFBP-1 to -4 were measured by immunoassays, as were pregnancy-associated plasma protein-A (PAPP-A) and the PAPP-A-generated N-terminal (NT) and C-terminal (CT) IGFBP-4 fragments, which are novel CVD risk markers. Arterial stiffness was evaluated by carotid-femoral pulse wave velocity (PWV). Cerebral white matter lesions (WMLs) and carotid artery remodelling were determined by MRI. RESULTS After multivariate adjustments, patients with T2D had lower concentrations of IGFBP-2, IGFBP-4, NT- and CT-IGFBP-4, when compared with controls. IGFBP-2 was inversely correlated to PWV in all subjects in multivariate analysis (P < 0.05), and IGFBP-3 was inversely associated with severity of WMLs (P < 0.05). The NT-IGFBP-4 fragment was associated with the degree of carotid artery remodelling among all subjects (regression coefficient (95% CI): 2.95 (0.70, 5.16), P = 0.011). Levels of NT- and CT-IGFBP-4 were reduced in T2D patients receiving metformin compared to those in controls and patients not receiving metformin. CONCLUSIONS Even in recently diagnosed and well-controlled T2D patients, IGF protein levels are altered and associated with CVD risk factors.

Fibroblast growth factor 21, fibroblast growth factor receptor 1, and β-Klotho expression in bovine growth hormone transgenic and growth hormone receptor knockout mice

OBJECTIVE Although growth hormone (GH) and fibroblast growth factor 21 (FGF21) have a reported relationship, FGF21 and its receptor, fibroblast growth factor receptor 1 (FGFR1) and cofactor β-Klotho (KLB), have not been analyzed in chronic states of altered GH action. The objective of this study was to quantify circulating FGF21 and tissue specific expression of Fgf21, Fgfr1, and Klb in mice with modified GH action. Based on previous studies, we hypothesized that bovine GH transgenic (bGH) mice will be FGF21 resistant and GH receptor knockout (GHR-/-) mice will have normal FGF21 action. DESIGN Seven-month-old male bGH mice (n=9) and wild type (WT) controls (n=10), and GHR-/- mice (n=8) and WT controls (n=8) were used for all measurements. Body composition was determined before dissection, and tissue weights were measured at the time of dissection. Serum FGF21 levels were evaluated by ELISA. Expression of Fgf21, Fgfr1, and Klb mRNA in white adipose tissue (AT), brown AT, and liver were evaluated by reverse transcription quantitative PCR. RESULTS As expected, bGH mice had increased body weight (p=3.70E-8) but decreased percent fat mass (p=4.87E-4). Likewise, GHR-/- mice had decreased body weight (p=1.78E-10) but increased percent fat mass (p=1.52E-9), due to increased size of the subcutaneous AT depot when normalized to body weight (p=1.60E-10). Serum FGF21 levels were significantly elevated in bGH mice (p=0.041) and unchanged in GHR-/- mice (p=0.88). Expression of Fgf21, Fgfr1, and Klb mRNA in white AT and liver were downregulated or unchanged in both bGH and GHR-/- mice. The only exception was Fgf21 expression in brown AT of GHR-/-, which trended toward increased expression (p=0.075). CONCLUSIONS In accordance with our hypothesis, we provide evidence that circulating FGF21 is increased in bGH animals, but remains unchanged in GHR-/- mice. Downregulation or no change in Fgf21, Fgfr1, and Klb expression are seen in white AT, brown AT, and liver of bGH and GHR-/- mice when compared to their respective controls, except for an increase in brown AT Fgf21 expression in GHR-/- mice, which could suggest a possible link to increased thermogenic potential in these mice. Overall, these results suggest possible modulation of FGF21 by GH resulting in FGF21 resistance or changes in FGF21 levels due to GH induced changes in liver size or kidney function.

PAPP-A, IGFBP-4 and IGF-II are secreted by human adipose tissue cultures in a depot-specific manner

OBJECTIVE Adipose tissue secretes pregnancy-associated plasma protein-A (PAPP-A), which may increase local IGF action through cleavage of IGF-binding protein-4 (IGFBP-4). We tested whether this mechanism was operational in human visceral and subcutaneous adipose tissue (i.e. VAT and SAT). DESIGN Explants of VAT and SAT from 26 obese subjects (hereof 17 women, BMI 39.5 (37.2; 42.8) kg/m2 (median (25%; 75% confidence interval) and SAT from eight lean, age-matched women (BMI 23.6 (22.4; 24.9) kg/m2) were incubated with or without GH (100 µg/L) and the media were harvested. METHODS Media were assessed for concentrations of PAPP-A, intact and PAPP-A-cleaved IGFBP-4, IGF-I and IGF-II, and IGF-I receptor (IGF-IR) activation by bioassay. RESULTS In obese subjects, VAT media contained higher concentrations than SAT of PAPP-A (4.4-fold) and both PAPP-A-generated IGFBP-4 fragments (C-terminal: 3.3-fold, N-terminal: 1.5-fold) (all P < 0.0005). Intact IGFBP-4 levels were similar in SAT and VAT. VAT media contained elevated IGF-II (1.4-fold; P < 0.005), but similar IGF-I concentrations compared with SAT. Still, VAT media contained a 1.8-fold increased ability to stimulate the IGF-IR (P < 0.005). IGF-I protein concentration and IGF-IR activation increased more in VAT media than SAT media following GH stimulation (both P < 0.05). At baseline, SAT media protein levels from lean and obese women were similar, with the exception of PAPP-A being 1.8-fold elevated in VAT media (P < 0.05). GH induced a similar increase in IGF-I media levels in SAT from obese and lean women. CONCLUSION Human adipose tissue cultures secrete enzymatically active PAPP-A, IGFBP-4 and IGF-II in a depot-specific manner, suggesting differential regulation of IGF activity. Further, IGF-II appears to be more prominent than IGF-I. Finally, VAT appears more GH responsive than SAT.

PAPP-A proteolytic activity enhances IGF bioactivity in ascites from women with ovarian carcinoma

Pregnancy-associated plasma protein-A (PAPP-A) stimulates insulin-like growth factor (IGF) action through proteolysis of IGF-binding protein (IGFBP)-4. In experimental animals, PAPP-A accelerates ovarian tumor growth by this mechanism. To investigate the effect of PAPP-A in humans, we compared serum and ascites from 22 women with ovarian carcinoma. We found that ascites contained 46-fold higher PAPP-A levels as compared to serum (P < 0.001). The majority (80%) of PAPP-A was enzymatically active. This is supported by the finding that ascites contained more cleaved than intact IGFBP-4 (P < 0.03). Ascites was more potent than serum in activating the IGF-I receptor (IGF-IR) in vitro (+31%, P < 0.05); in 8 of 22 patients by more than two-fold. In contrast, ascites contained similar levels of immunoreactive IGF-I, and lower levels of IGF-II (P < 0.001). Immunohistochemistry demonstrated the presence of IGF-IR in all but one tumor, whereas all tumors expressed PAPP-A, IGFBP-4, IGF-I and IGF-II. Addition of recombinant PAPP-A to ascites increased the cleavage of IGFBP-4 and enhanced IGF-IR activation (P < 0.05). In conclusion, human ovarian tumors express PAPP-A, IGFBP-4 and IGFs and these proteins are also present in ascites. We suggest that both soluble PAPP-A in ascites and tissue-associated PAPP-A serve to increase IGF bioactivity and, thereby, to stimulate IGF-IR-mediated tumor growth.

Insulin-Like Growth Factor Bioactivity, Stanniocalcin-2, Pregnancy-Associated Plasma Protein-A, and IGF-Binding Protein-4 in Pleural Fluid and Serum From Patients With Pulmonary Disease

Context: Members of the insulin‐like growth factor (IGF) system are primarily produced in the liver and secreted into the circulation, but they are also produced, recruited, and activated locally in tissues. Objective: To compare activity and concentrations of IGF system components in pleural fluid and blood. Design: Pathological pleural fluid, secondary to lung cancer or nonmalignant disease, and matching blood samples were collected from 24 patients ages 66.7 to 81.9 years. Methods: IGF‐related proteins and cytokine levels were measured by immunoassays or immunoblotting. Bioactive IGF was measured by an IGF‐1 receptor phosphorylation assay. Results: Total IGF‐1 concentration did not differ between the compartments, but concentrations of free IGF‐1 and bioactive IGF were more than threefold higher in pleural fluid than in corresponding serum samples (P = 0.0004), regardless of etiology. Median pregnancy‐associated plasma protein‐A (PAPP‐A) and interleukin (IL)‐6 levels were increased 47‐fold and 143‐fold, respectively, in pleural fluid compared with plasma (P < 0.0001). PAPP‐A and IL‐6 concentrations correlated positively (r = 0.46; P = 0.02). In pleural fluid, levels of PAPP‐A‐generated IGF binding protein‐4 fragments correlated inversely with that of stanniocalcin‐2 (r ≤ ‐0.42; P ≤ 0.05), a PAPP‐A inhibitor; such correlations were absent in plasma. Conclusion: Pathological pleural fluid is characterized by increased in vitro IGF bioactivity and elevated concentrations of PAPP‐A, an IGF‐activating proteinase. Thus, the tissue activity of the IGF system may differ substantially from that of the circulating IGF system. The correlation between IL‐6 and PAPP‐A indicates that inflammation plays a role in promoting local tissue IGF activity.