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Title: Implication of pappalysins and stanniocalcins in the bioavailability of IGF-I in children with

type 1 diabetes mellitus

Authors:

María Güemes

, Álvaro Martín-Rivada

, Beatriz Corredor

, Patricia Enes

, Sandra Canelles

Vicente Barrios

1, 2

, Jesús Argente

1, 2, 3,4

Affiliations:

Hospital Infantil Universitario Niño Jesús, Departments of Pediatrics & Pediatric Endocrinology,

Research Institute “La Princesa,”

Centro de Investigación Biomédica en Red de Fisiopatología de la

Obesidad y Nutrici

n (CIBEROBN), Instituto de Salud Carlos III, E-28029, Madrid, Spain.

Department

of Pediatrics, Universidad Autónoma de Madrid, E-28029, Madrid, Spain.

IMDEA, Food Institute,

CEIUAM+CSI, Cantoblanco, E-28049, Madrid, Spain.

Key Words:

Type 1 diabetes mellitus, growth, IGF-I, IGFBP, pappalysin, stanniocalcin, children

Corresponding author´s contact information:

Prof. Dr. Jesús Argente

Hospital Infantil Universitario Niño Jesús, Departments of Pediatrics & Pediatric Endocrinology

Avenida Menéndez Pelayo 65. 28009 Madrid. Spain

Email address:

jesus.argente@fundacionendo.org

ORCID #: 0000-0001-5826-0276

Grants or fellowships supporting this manuscript

This research was funded by the Ministerio de Ciencia e Innovación (FIS-PI19/00166 to JA), and by

“ERDF A way of making Europe”, the “European Union”, and Centro de Investigación Biomédica en

Red Fisiopatología de Obesidad y Nutrición (CIBEROBN), Instituto Carlos III (JA). This research was

also funded by a grant from

“2019 Proyectos intramurales. Carrera Corre por el Niño”

awarded by

Fundación para la Investigación Biomédica (FIB) Hospital Infantil Universitario Niño Jesús, Madrid,

Spain (JA, MG). The rest of the authors received no funding related to this project.

Disclosure information:

The authors have no conflict of interest to declare.

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ABSTRACT

INTRODUCTION:

Anomalies in the growth hormone (GH) - insulin-like growth factor (IGF) axis, are

common in children with type 1 diabetes mellitus (T1DM), even in those reaching a normal or near-

normal final height. However, concentrations of the IGF bioavailability regulatory factors [pappalysins

(PAPP-As) and stanniocalcins (STCs)] have not been reported in children with T1DM.

AIMS:

To determine serum concentrations of PAPP-As and STCs in children at diagnosis of T1DM and

after insulin treatment and the correlation of these factors with other members of the GH/IGF, beta-

cell insulin reserve, auxology and nutritional status.

METHODS:

A single-center prospective observational study including 47 patients (59.5% males), with

T1DM onset at median age of 9.2 (IQR: 6.3, 11.9) years was performed. Blood and anthropometric

data were collected at diagnosis and after 6 and 12 months of treatment.

RESULTS:

At 6 and 12 months after T1DM diagnosis, there was improvement in the metabolic control

[decrease in HbA1c at 12 months -3.66 CI95% (-4.81,-2.05), p=0.001], as well as in body mass index

SD and height SD (not statistically significant). STC2 increased (p<0.001) and PAPP -A2 decreased

(p<0.001) at 6 and 12 months of treatment onset (p<0.001), which was concurrent with increased

total IGF-I as well and IGFBP concentrations, with no significant modification in free IGF -I

concentrations. HbA1c correlated with PAPPA-2 (r: +0.41; P<0.05) and STC2 (r: -0.32; P<0.05).

CONCLUSIONS:

Implementation of insulin treatment after T1DM onset modifies various components

of the circulating IGF system, including PAPP-A2 and STC2. How these modifications modulate linear

growth remains unknown.

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INTRODUCTION

Although significant advancements in insulin therapy for children with type 1 diabetes mellitus

(T1DM) have allowed many patients to reach normal or just slightly reduced final height, anomalies

of growth often persist (

). Suboptimal pre- and pubertal growth likely reflects the duration of the

disease and metabolic control (

2, 3

), as well as the impact of inflammatory markers, including

interleukin-6, C-reactive protein, and fibrinogen that directly affect the growth plate and suppress

local insulin-like growth factor (IGF-I) actions (

4, 5

). Uncontrolled celiac disease and potentially, the

recently popular very low carbohydrate diets if not carefully managed

(6)

can also affect growth. To

ensure physiological growth, normal insulin secretion and, especially, its portal concentrations, is

indispensable for normal circulating concentrations of IGF-I and IGF-binding proteins (IGFBPs) (

Insulin is also reported to influence hepatic expression of the GH receptor and to participate in post -

receptor GH signaling, which influences IGF-I and IGFBP synthesis (

). Low IGF-I concentrations lead

to a reduction in its negative feedback on the pituitary, which is involved in the observed GH

hypersecretion (

). Exogenous insulin administered subcutaneously, or even via continuous infusion,

is not able to ameliorate portal hepatic hypoinsulinization (

9, 10

). Insufficient intraportal insulin in

children with T1DM results in low circulating concentrations of IGF-I and IGFBP-3, as well as high

IGFBP-1 (

) and GH concentrations (

). Additionally, high IGFBP-1 levels could also inhibit IGF-I

bioactivity (

The growth regulatory factors [pappalysins (PAPP-A and PAPP-A2) and stanniocalcins (STC1 and

STC2)] modulate the bioavailability of IGFs by regulating the concentrations of intact and free IGFBPs.

In the circulation, IGFBPs bind IGF-I or IGF-II and acid-labile subunit (ALS) forming trimolecular

complexes, and thus antagonizing the binding of free forms to the receptor. Pappalysins cleave

IGFBPs, and hence can augment IGF bioactivity and subsequently enhance local and systemic IGF

signaling (

The main target of PAPP-A is IGFBP-4

(13)

, but it can also cleave IGFBP-5

(14)

and

IGFBP-2

(15)

to a lesser extent. Alternatively, PAPP-A2 cleaves IGFBP-3, as well as IGFBP-5

(14)

, and

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IGFBP-2

(16)

, and is considered one of the principal regulators of IGF-I bioavailability

(17).

Stanniocalcins are glycoproteins known to inhibit the action of pappalysins (

18, 19

Concentrations of pappalysins and stanniocalcins have not yet been determined in children with

T1DM, albeit their study in adults with diabetes has recently gained interest

(20, 21, 22)

Stanniocalcins are expressed in the pancreatic islets, where STC1 colocalizes with insulin in beta-cells

(

) and STC2 with glucagon in alpha-cells (

), where they are suggested to exert effects on glucose

homeostasis and to be markers of the appearance and progression of diabetes (

21, 22

). In a 20-year

longitudinal study with 1506 participants, after multivariable selection, PAPP-A was the only protein

associated with the onset of pre-diabetes and type 2 diabetes (T2DM), with associations still

observed at the 20-year visit (

). Another study in adults with T2DM, following multivariable

adjustment, found that higher concentrations of STC2, PAPP-A and both intact and total IGFBP-4

associated with all-cause mortality

(24)

Conversely, other studies have not found a consistent

association between PAPP-A and T2DM (

25, 26, 27

)

With the aim to better characterize the physiology of growth in children with T1DM, this study

investigated: 1) The baseline serum concentrations of pappalysins and stanniocalcins upon diagnosis;

2) The effect of insulin treatment on these factors; 3) The possible correlation with members of the

GH/IGF axis, beta-cell insulin reserve, auxology and nutrition status.

PATIENTS AND METHODS

Ethics statement:

This study was approved by the Ethical committee of

Hospital Infantil Universitario

Niño Jesús, and adheres to the ethical principles of the Declaration of Helsinki.

Patients and methods:

This single-center prospective observational study was carried out in pediatric

patients (age < 18 years) diagnosed with T1DM. Positive pancreatic autoimmunity was an inclusion

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criterion. Forty-seven children were assessed between November 2019 and November 2021. The

study was carried out in three stages: upon diagnosis during hospitalization (T0), 6 months after

diagnosis (T1) and 12 months after diagnosis (T2). See Figure 1 where the flowchart of data

collection is depicted.

Demographics (sex, date of birth) and medical conditions were recorded. Patients were clinically

evaluated at the endocrinology department where weight, body mass index (BMI) and standing

height measurements were performed at T0, T1 and T2, with growth velocity being assessed at T1

and T2, and standardized according to Spanish normative data (

). Pubertal status was assessed by

Tanner staging.

Serum analysis:

Fasting blood samples were extracted at T0, T1 and T2. Total and free IGF-I, IGF-II,

total IGFBP-2 and -5, intact and total IGFBP-3 and -4, PAPP-A, and STC2 were determined by using

commercial ELISA kits (Ansh Labs, Webster, TX, USA). STC1 was measured by ELISA (R&D Systems,

Minneapolis, MN, USA). ALS was measured by ELISA (Mediagnost, Reutlingen, Germany), and PAPP -

A2 was determined by a chemiluminescence immunoassay (Cloud Clone, Katy, TX, USA). Research

Resource Identifiers for the immunoassays employed in this study are available in Table 1. All data of

the IGF axis were standardized for sex and pubertal development, according to recent normative

data (

). Capillary HbA1c was analyzed in the outpatient clinic (DCA Vantage Analyzer Class 1,

Siemens Healthcare Diagnostics Ltd, Camberley, UK). Glucose, insulin (this parameter was only

analyzed at T0), C-peptide, total T3, transferrin, prealbumin and retinol binding protein were

analyzed in the hospital’s

validated laboratory. Metabolic control determined by means of time in

range as measured by a glucose sensor was unavailable at the time in many patients, and thus, not

included in this study.

Statistics:

Quantitative variables are expressed as median and interquartile range (IQR). Qualitative

variables are expressed as absolute and relative frequencies. To check for normality, the Shapiro -Wilk

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test was used. Differences between groups were investigated with ANOVA for repeated measures.

Logarithmic transformation was used to transform skewed variables into a normalized dataset. The

Fisher correlation test was performed to determine whether there was linear association between

quantitative variables. The statistical analysis was performed with STATA 15.1. Results were

considered statistically significant when the P value was below 0.05.

RESULTS

Patients

The median age of diabetes onset was 9.16 years old (IQR: 6.30, 11.91), with twenty -eight males

(59.5%) and nineteen females (40.4%).

They were all previously healthy children except for 3 cases

with autoimmune thyroiditis, 1 with celiac disease (adhering to gluten-free diet at diagnosis of T1DM

and throughout this study) and one case with 17q12 microdeletion. None of them were receiving

chronic medications whilst in this study. Twelve cases (25.5%) had a positive family history of T1DM.

Autoantibodies (anti-GAD +/- anti-insulin +/- anti-IA2 +/- ICA) were positive in all cases.

Metabolic control, growth and nutrition

With regards to the onset of the disease, 16 children (34%) presented with severe ketoacidosis, 9

(19.1%) with moderate ketoacidosis, 9 (19.1%) with mild ketoacidosis and 13 (27.6%) with

hyperosmolar hyperglycemia. The median HbA1c at diagnosis was 11.2% (IQR: 9.8, 13.1). At T1,

median C-peptide was 0.23 ng/ml (IQR: 0.17, 0.57) and HbA1c 7.2% (IQR: 6.7, 8.2). At T2, median C-

peptide was 0.16 ng/ml (IQR: 0.05, 0.41) and HbA1c 7.6% (IQR: 6.8, 8.5). There was a statistically

significant improvement in HbA1c from T0 to T1 and T2 (Table 2), whereas no statistical significance

was found in C-peptide change.

At the time of diagnosis of T1DM (T0), the median patient height was -0.44 SD (IQR: -0.35, 1.05),

weight was -0.1 SD (IQR: -0.64, 0.54), BMI was -0.27 SD (IQR: -1.11, 0.20) and Tanner stages were: I

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(29/47); II (6/47); III (5/47); IV (2/47) and V (5/47). At T1, median height was 0.05 SD (IQR: -0.30,

0.86), weight -0.12 SD (IQR: -0.62, 0.42), BMI -0.03 SD (IQR: -0.74, 0.3). At T2, median height was 0.0

SD (IQR: -0.10, 1.04), weight -0.10 SD (IQR: -0.67, 0.76), BMI 0.0 SD (IQR: -0.78, 0.3). There was no

statistical difference between height, weight and BMI SD from T0, to T1 and T2 (Table 2). Growth

velocity SD at T1 was 0.80 (IQR: -0.40, 1.79) and at T2 0.70 (IQR: 0.33, 1.00), with no statistically

significant difference (Table 3).

All patients confirmed that they followed a varied Mediterranean diet prior and during this study.

Albeit nutritional markers were not extracted at T0, available data at T1 shows a median transferrin

of 244 mg/dl (IQR: 219, 263), prealbumin 16.8 mg/dl (IQR: 16.1, 19), retinol binding globulin 1.32

mg/dl (IQR: 1.06, 1.51) and T3T 1.36 ng/ml (IQR: 1.26, 1.49). At T2, median transferrin was 267

mg/dl (IQR: 258, 284), prealbumin 18.6 mg/dl (IQR: 17.0, 20.8), retinol binding globulin 1.26 mg/dl

(IQR: 1.15, 1.41) and T3T 1.38 ng/ml (IQR: 1.23, 1.51). Transferrin was the only nutritional marker

which significantly increased from T1 to T2 (Table 3).

Serum concentrations of the GH/IGF axis

All serum concentrations of pappalysins, stanniocalcins, free and total IGF-I, IGF-II, and total and

intact IGFBPs are presented in Figure 2 and Table 4. There were no significant differences in

concentrations between both sexes. All analyte concentrations remained within the limits of

normality (-2.0, +2.0 SD) during this study, except for an outlier value for total IGF-II at T2 [median:

1.71 SD (IQR: 1.08, 2.41)] and IGFBP-5 at T2 [1.95 SD (0.72, 2.42)]. However, upon diagnosis, STC2

concentrations were in the lower part of normality [-1.31 SD (-1.97, -0.39)] and significantly

increased after 6 and 12 months of insulinization [0.07 SD (-0.37, 0.69) p<0.001 and 0.52 SD (-0.76,

0.90) p<0.001, respectively]. The STC2 increase was paralleled by a significant decrease in PAPPA-A2.

PAPPA-A2 concentrations were in the upper part of normality at diagnosis [1.05 SD (0.68, 1.63)], and

significantly decreased during insulinization [6 months: 0.49 SD (-0.07, 0.92) p<0.001; 12 months:

0.13 SD (-0.16, 0.52) p<0.001]. STC1 and PAPP-A concentrations were within their normal range at

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diagnosis and remained unvaried throughout the study. Total and free IGF-I SD and IGFBP-3

concentrations increased during insulin treatment, but remained within the normal range at all

times. There was also an increase in total IGF-II, total IGFBP-3, total IGFBP-4, IGFBP-5, as well as in

intact IGFBP-3, intact IGFBP-4, ALS and PAPP-A over time. IGFBP-2 concentrations remained unvaried.

When investigating for differences between variables depending on Tanner stage (prepubertal versus

pubertal stages), total IGF-II showed statistical change in prepubertal children [Delta mean T1 vs T0:

0.97 CI95% (-0.35, 2.28) P= 0.026; Delta mean T2 vs T0 2.22 CI95% (0.58, 3.85) P= 0.009]. No other

variable showed statistical difference between pre and pubertal stage.

Correlations

Correlations were examined between pappalysins, stanniocalcins and markers of the growth axis

(IGFs, IGFBPs), beta-cell reserve (C-peptide), initial presentation (ketoacidosis, hyperglycemia),

HbA1c, exogenous insulin dose, auxology (height, growth velocity, weight, BMI) and nutrition

(prealbumin, transferrin, T3T and retinol). Significant correlations are collected in Table 5. There were

no significant correlations found with growth velocity, C-peptide, initial presentation and insulin

dose.

DISCUSSION

To the best of our knowledge, this is the first study to report circulating concentrations of

pappalysins and stanniocalcins in children with T1DM. As expected, upon exogenous insulinization

there was significant metabolic (HbA1c) and nutritional improvement (transferrin) during the

following year. Other parameters showed a tendency to improvement, such as growth velocity and

BMI, although this was not statistically significant, probably because they were already within the

normal range at presentation. The impact of height at T1DM diagnosis has been much debated, with

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studies reporting children with T1DM being taller than their healthy peers (

1, 30

), but this was not

confirmed by other studies (

). One suggested explanation for the increased height of children at

the onset of T1DM, or “accelerator” hypothesis (

1, 30

), involves the insulinopenia in the prediabetic

phase that results in increased IGFBP-3 proteolysis, with a subsequent rise in the availability of IGF-I

(

). Herein, we confirm this increase in IGFBP-3 proteolysis; however, no relevant changes in

circulating free IGF-I were observed, likely due to the data dispersion. In contrast to a previous study,

we found no association between growth velocity after T1DM onset and beta-cell reserve (evaluated

by C-peptide concentrations after treatment) (

). A possible explanation for this is that most of our

patients already had a normal stature at presentation and their growth velocity did not statistically

differ after insulinization. Total IGF-I concentrations were lowest at diagnosis, when the highest

HbA1c levels were obtained. This is in accordance with other studies in pediatric populations (

11,

), as well as the increase in free IGF-I over time following metabolic improvement with intensive

exogenous insulin treatment (

Significant modifications in STC2 levels were found during the study period, but not in STC1. We

previously hypothesized that there is a more potent role of STC2 versus STC1 with regards to impact

on linear bone growth (

), with STC2 also possessing other physiological roles in calcium-phosphate

regulation, cell development, cytoprotection, and angiogenesis

(25, 34)

, as well as affecting appetite

and body weight regulation

(34, 35)

. Although a correlation was found between STC2 and body fat

percentage in one cross-sectional human study (

), we observed no correlation with body mass

index, although there was a significant correlation with prealbumin, suggesting nutritional

implication. The relationship between serum concentrations of STC2 and glucose metabolism

remains uncertain. A study of 122 healthy adults failed to observe a relationship between these

factors (

), which is similar to another study in adult T2DM patients (

21)

. However, another study in

adults with T2DM found that those with the highest HbA1c exhibited the lowest expression of STC2

in platelets (

). Moreover, in post Roux-en-Y adults those with a decrease in STC2, the levels of total

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IGFBP-4 correlated with an improvement in HbA1c, fasting glucose and insulin (

). The decrease in

PAPP-A2 over time coincides with the increase of most total forms of IGF, IGFBPs and ALS, with no

statistical change in the free forms (Figure 3 schematically represents this study´s findings). In

concordance with these findings, HbA1c negatively correlated with STC2 and positively with PAPPA -2.

Metabolic improvement over time associates with an increase in the total forms of IGF-I and IGFBP-3,

and coincides with the described changes in STC2 and PAPP-A2. The underlying pathophysiological

explanation of this remains unclear.

Several total forms of IGFBPs increased overtime, including IGFBP-5, which is known to have a role in

muscle growth and differentiation

(37)

. Unfortunately, discrepancies between IGFBP measurements

across studies often occur as many assays are not able to reliably differentiate between intact and

degraded IGFBP. IGFBP-2 is reported to participate in growth regulation, body composition, bone

development

(38)

and to have antidiabetic effects

(39)

; however, we found no relevant modifications

in its concentrations during the first year of insulin treatment or in comparison with the

concentrations in healthy children; even though it has been described to be inhibited by insulin (

The association between PAPP-A and diabetes is inconsistent in clinical studies in adults, with

statistical association between PAPP-A and pre-diabetes and T2DM reported in one study (

), but

not in others (

25, 26, 27

)

. During maintained hyperglycemia, TGF-

β is reported to upregulate the

expression of PAPP-A and IGF signaling

(19)

. However, here PAPP-A levels were not significantly

increased and did not show relevant modifications over time. An explanation for this could be, that

PAPP-A has an active proteolytic action at the cellular level

(15)

, in proximity to the IGF-I receptor

(IGF-IR) in various target tissues, and therefore mainly influences IGF-I actions locally

(15)

, with

concentrations changing at the tissue level. Furthermore,

PAPP-A concentrations may not represent

modifications in its proteolytic activity, which has been previously reported to change under different

physiological situations

(41)

. It is also possible that increased levels of STC2 inhibit PAPP-

A’s activity

(42)

. Furthermore, our previous pediatric study showed that PAPP-A concentrations remain fairly

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constant during postnatal life in healthy children

(29)

, as they appear to do during the first year after

T1DM onset. Unlike in healthy individuals where no correlation was found between concentrations

of STC1 and PAPP-A

(29)

, correlation was identified in T1DM, of uncertain explanation given the small

change overtime of the concentrations of both analytes.

IGFBP-4 is known to have roles in skeletal growth

(43)

bone physiology

(44)

and, in children with

obesity it positively correlates with fasting insulin concentrations

(45)

. Here, total IGFBP-4

significantly increased at 12 months, in unison with the rise in STC2, despite no significant

modification in PAPP-A levels. PAPP-A was formerly considered the only protease to degrade IGFBP-4

(

), albeit metalloproteases other than the PAPP-As proteolyze IGFBPs (

). This supports the

hypothesis that at a cellular level STC2 blocks the degradation of IGFBP-4 through inhibition of PAPP-

A activity (

Although PAPP-A, IGFBP-4 and STC2 are expressed in adipose tissue and are known to change with

weight loss (

25, 34

), here no specific association with BMI was found. However, a negative

correlation of IGFBP-4 with both prealbumin and retinol binding protein was found, while STC2

correlated with the nutritional marker prealbumin suggesting a nutritional role of these factors.

Interestingly, no statistical differences were observed between sexes or when comparing prepubertal

and pubertal concentrations of all the studied markers of the GH/IGF axis. The exception to the latter

involves total IGF-II concentrations that were significantly higher in prepubertal versus pubertal

children, which is in contrast to a study where no changes were found during childhood

(48)

. This

discrepancy is not yet fully understood.

Given that STC1 colocalizes with insulin in beta-cells (

), its suitability as a diabetes marker has been

considered (

21, 22

). However, we found minimal changes in its concentrations during follow -up,

suggesting that it may not serve as a beta-cell reserve marker in children with T1DM. In adult T2DM

patients, significant correlation was found between STC1, glycemia and HbA1c

(22)

, but not here in

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our study of T1DM. This lack of correlation is logical as in T1DM the insulin reserves are basically

non-existent as opposed to T2DM.

The main caveat of this study includes only measurement of circulating concentrations, as

quantifying the activity of pappalysins and stanniocalcins in serum or at the cellular level (

) would

provide further in-depth understanding of the physiology of the peripheral GH/IGF axis in these

patients. However, we have recently published circulating levels of members of this axis in patients

with anorexia nervosa and Prader-Willi syndrome (

49, 50)

, with these studies indicating that indeed

information on circulating levels can provide relevant information. Another limitation of this study is

the lack of growth velocity prior to the diagnosis of T1DM and before starting insulin therapy (during

a period of insulinopenia), to confirm the clinical implications of the findings in this study. Moreover,

if two groups had been available (one poorly controlled and one with good glycemic control) to

compare the circulating IGF system in both groups, further insight regarding the importance of

glycemic control may have been concerned in the study; however, all patients had optimal metabolic

control during the duration of the study. During the first year after T1DM diagnosis most families

become highly implicated in the correct management of diabetes and, under these circumstances,

growth should be minimally impacted. Thus, a more long-term study may be of interest as often,

years after the onset of the disease when patients and/or relatives/caregivers become less strict with

metabolic control and this could impact growth. Additionally, the growth-promoting effect of insulin

through the ubiquitously expressed IGF-IR (

), as well as IGF-IR signaling promoting insulin

sensitivity (

), should be considered as possible growth enhancers.

CONCLUSIONS

Our results indicate that implementation of insulin treatment after T1DM onset modifies various

components of the circulating IGF system, including those of PAPP-A2 and STC2. The orchestrated

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interplay between the GH/IGF members, along with adequate metabolic control and nutrition

optimization, due to correct insulinization, participate in the promotion of linear growth.

ACKNOWLEDGEMENTS

The authors would also like to extend their gratitude to Dr Purificación Ros for her contribution to

this study.

DATA AVAILABILTY

Some or all datasets generated during and/or analyzed during the current study are not publicly

available but are available from the corresponding author on reasonable request.

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REFERENCES

1- Bizzarri C, Timpanaro TA, Matteoli MC, Patera IP, Cappa M, Cianfarani S. Growth Trajectory in

Children with Type 1 Diabetes Mellitus: The Impact of Insulin Treatment and Metabolic Control.

Horm Res Paediatr. 2018;89(3):172-177.

2- Marcovecchio ML, Heywood JJ, Dalton RN, Dunger DB. The contribution of glycemic control to

impaired growth during puberty in young people with type 1 diabetes and microalbuminuria. Pediatr

Diabetes. 2014;15(4):303

–

308.

3- Waden J, Forsblom C, Thorn LM, et al. Adult stature and diabetes complications in patients with

type 1 diabetes: the FinnDiane Study and the diabetes control and complications trial. Diabetes.

2009; 58(8):1914

–

1920.

4- Snell-Bergeon JK, West NA, Mayer-Davis EJ, et al. Inflammatory markers are increased in youth

with type 1 diabetes: the SEARCH Case-Control study. J Clin Endocrinol Metab. 2010; 95(6): 2868

–

2876.

5- Sederquist B, Fernandez-Vojvodich P, Zaman F, Savendahl L. Recent research on the growth plate:

Impact of inflammatory cytokines on longitudinal bone growth. J Mol Endocrinol. 2014;53(1):35

–

44.

6- Koren D. Growth and development in type 1 diabetes. Curr Opin Endocrinol Diabetes Obes.

2022;29(1):57-64.

7- Dunger DB, Cheetham TD. Growth hormone insulin-like growth factor I Axis in insulin-Dependent

diabetes mellitus. Hormone Research. 1996; 46: 2

–

8- Clark PA, Clarke WL, Pedadda S, et al. The effects of pubertal status and glycemic control on the

growth hormone-IGF-I axis in boys with insulin-dependent diabetes mellitus. J Pediatr Endocrinol

Metab. 1998;11:427-435.

9- Shishko PI, Dreval AV, Abugova IA, Zajarny IU, Goncharov VC. Insulin-like growth factors and

binding proteins in patients with recent- onset type 1 (insulin-dependent) diabetes mellitus:

ACCEPTED MANUSCRIPT

Downloaded from https://academic.oup.com/jes/advance-article/doi/10.1210/jendso/bvae081/7656891 by UCSD-Philosophy user on 23 April 2024

influence of diabetes control and intraportal insulin infusion. Diabetes Research and Clinical Practice.

1994; 25: 1

–

12.

10- Bizzarri C, Benevento D, Giannone G, et al. Sexual dimorphism in growth and insulin-like growth

factor-I in children with type 1 diabetes mellitus. Growth Hormone and IGF Research. 2014; 24:256

–

259.

11- Muñoz MT, Barrios V, Pozo J, Argente J. Insulin-like growth factor I, its binding proteins 1 and 3,

and growth hormone-binding protein in children and adolescents with insulin-dependent diabetes

mellitus: Clinical implications. Pediatr Res. 1996; 39: 992

–

998.

12- Oxvig C, Conover CA. The Stanniocalcin-PAPP-A-IGFBP-IGF Axis. J Clin Endocrinol Metab. 2023;

16;108(7):1624-1633.

13- Lawrence JB, Oxvig C, Overgaard MT, et al. The insulin-like growth factor (IGF)-dependent IGF-

binding protein-4 protease secreted by human fibroblasts is pregnancy-associated plasma protein-A.

Proc Natl Acad Sci U S A. 1999;96(6):3149-3153.

14- Laursen LS, Overgaard MT, Søe R, et al. Pregnancy-associated plasma protein-A (PAPP-A) cleaves

insulin-like growth factor binding protein (IGFBP)-5 independent of IGF: implications for the

mechanism of IGFBP-4 proteolysis by PAPP-A. FEBS Lett. 2001;504(1-2):36-40.

15- Monget P, Mazerbourg S, Delpuech T, et al. Pregnancy-associated plasma protein-A is involved in

insulin-like growth factor binding protein-2 (IGFBP-2) proteolytic degradation in bovine and porcine

preovulatory follicles: identification of cleavage site and characterization of IGFBP-2 degradation. Biol

Reprod. 2003;68(1):77-86.

16- Russo VC, Azar WJ, Yau SW, Sabin MA, Werther GA. IGFBP-2: the dark horse in metabolism and

cancer. Cytokine and Growth Factor Reviews. 2015;26:329

–

346.

17- Barrios V, Chowen JA, Martín-Rivada Á, et al. Pregnancy-associated plasma protein (PAPP)-A2 in

physiology and disease. Cells. 2021;10(12):3576.

ACCEPTED MANUSCRIPT

Downloaded from https://academic.oup.com/jes/advance-article/doi/10.1210/jendso/bvae081/7656891 by UCSD-Philosophy user on 23 April 2024

18- Kløverpris S, Mikkelsen JH, Pedersen JH, et al. Stanniocalcin-1 potently inhibits the proteolytic

activity of the metalloproteinase pregnancy-associated plasma protein-A. J Biol Chem. 2015;

290:21915

–

21924.

19- Jepsen MR, Kløverpris S, Mikkelsen JH, et al. Stanniocalcin-2 inhibits mammalian growth by

proteolytic inhibition of the insulin-like growth factor axis. J Biol Chem. 2015;290: 3430

–

3439.

20- Zaidi D, Turner JK, Durst MA, et al. Stanniocalcin-1 co-localizes with insulin in the pancreatic

islets. ISRN Endocrinol. 2012; 2012: 834359.

21- Moore EE, Kuestner RE, Conklin DC, et al. Stanniocalcin 2: characterization of the protein and its

localization to human pancreatic alpha cells. Horm Metab Res. 1999; 31: 406

–

414.

22- López JJ, Jardín I, Cantonero Chamorro C, et al. Involvement of stanniocalcins in the deregulation

of glycaemia in obese mice and type 2 diabetic patients. J Cell Mol Med. 2018;22(1):684-694.

23- Ferreira JP, Lamiral Z, Xhaard C, et al. Circulating plasma proteins and new-onset diabetes in a

population-based study: proteomic and genomic insights from the STANISLAS cohort. European

Journal of Endocrinology. 2020; 183(3):285-295.

24- Gude MF, Hjortebjerg R, Bjerre M, et al. The STC2-PAPP-A-IGFBP4-IGF1 axis and its associations to

mortality and CVD in T2D. Endocr Connect. 2023; 11:12(3):e220451.

25- Hjortebjerg R. Metabolic improvement after gastric bypass correlates with changes in IGF -

regulatory proteins stanniocalcin-2 and IGFBP-4. Metabolism Clinical and Experimental. 2021;124

:154886.

26- Pellitero S, Reverter JL, Pizarro E, et al. Pregnancy-associated plasma protein-a levels are related

to glycemic control but not to lipid profile or hemostatic parameters in type 2 diabetes. Diabetes

Care. 2007; 30(12): 3083-3085.

27- Aso Y, Okumura K, Wakabayashi S, Takebayashi K, Taki S, Inukai T. Elevated pregnancy-associated

plasma protein-a in sera from type 2 diabetic patients with hypercholesterolemia: associations with

carotid atherosclerosis and toe-brachial index. J Clin Endocrinol Metab. 2004; 89 (11): 5713-5717.

ACCEPTED MANUSCRIPT

Downloaded from https://academic.oup.com/jes/advance-article/doi/10.1210/jendso/bvae081/7656891 by UCSD-Philosophy user on 23 April 2024

28- Hernández M, Castellet J, Narvaiza JL, et al. Curvas y Tablas de Crecimiento. Instituto de

Investigación sobre Crecimiento y Desarrollo; 1988.

29- Martín-Rivada Á, Guerra-Cantera S, Campillo-Calatayud A, et al. Pappalysins and Stanniocalcins

and Their Relationship With the Peripheral IGF Axis in Newborns and During Development. J Clin

Endocrinol Metab. 2022; 28:107(10):2912-2924.

30- Dahlquist G. Can we slow the rising incidence of childhood-onset autoimmune diabetes? The

overload hypothesis. Diabetologia. 2006; 49(1):20

–

24.

31- Cianfarani S, Bonfanti R, Bitti ML, et al. Growth and insulin-like growth factors (IGFs) in children

with insulin-dependent diabetes mellitus at the onset of disease: Evidence for normal growth, age

dependency of the IGF system alterations, and presence of a small (approximately 18-kilodalton) IGF-

binding protein-3 fragment in serum. J Clin Endocrinol Metab. 2000; 85: 4162

–

4167.

32- Raisingani M, Preneet B, Kohn B, Yakar S. Skeletal growth and bone mineral acquisition in type 1

diabetic children; abnormalities of the GH/IGF-1 axis. Growth Hormone and IGF Research. 2017;34:

–

21.

33- Bereket A, Lang CH, Blethen SL, Ng LC, Wilson TA. Insulin treatment normalizes reduced free

insulin-like growth factor-I concentrations in diabetic children. Clin Endocrinol. (Oxf.) 1996;45:321

–

326.

34- Argente J, Chowen JA, Pérez-Jurado LA, Frystyk J, Oxvig C. One level up: abnormal proteolytic

regulation of IGF activity plays a role in human pathophysiology. EMBO Mol Med. 2017;9(10):1338-

1345.

35- Jiao Y, Zhao J, Shi G, et al. Stanniocalcin2 acts as an anorectic factor through activation of STAT3

pathway. Oncotarget. 2017;8:91067

–

91075.

36- Panagiotou G, Anastasilakis AD, Kynigopoulos G, et al. Physiological parameters regulating

circulating levels of the IGFBP-4/Stanniocalcin-2/PAPP-A axis. Metabolism. 2017;75:16

–

24.

ACCEPTED MANUSCRIPT

Downloaded from https://academic.oup.com/jes/advance-article/doi/10.1210/jendso/bvae081/7656891 by UCSD-Philosophy user on 23 April 2024

37- Duan C, Ren H, Gao S. Insulin-like growth factors (IGFs), IGF receptors, and IGF-binding proteins:

roles in skeletal muscle growth and differentiation. Gen Comp Endocrinol. 2010;167(3):344-351.

38- Russo VC, Azar WJ, Yau SW, Sabin MA, Werther GA. IGFBP-2: the dark horse in metabolism and

cancer. Cytokine Growth Factor Rev. 2015;26(3):329-346.

39- Hedbacker K, Birsoy KV, Wysocki RW, et al. Antidiabetic effects of IGFBP2, a leptin -regulated

gene. Cell Metab. 2010;11(1):11-22.

40- Brynskov T, Laugesen CS, Floyd AK, Frystyk J, Sørensen TL. The IGF-Axis and diabetic retinopathy

before and after gastric bypass surgery. Obes Surg. 2016:1

–

41- Gyrup C, Christiansen M, Oxvig C. Quantification of proteolytically active pregnancy-associated

plasma protein-A with an assay based on quenched fluorescence. Clin Chem. 2007;53(5):947-954.

42- Kobberø SD, Gajhede M, Mirza OA, et al. Structure of the proteolytic enzyme PAPP -A with the

endogenous inhibitor stanniocalcin-2 reveals its inhibitory mechanism. Nat Commun. 2022;

18:13(1):6084.

43- Maridas DE, DeMambro VE, Le PT, et al. IGFBP-4 regulates adult skeletal growth in a sex-specific

manner. J Endocrinol. 2017;233(1):131-144.

44- Ranke MB. Insulin-like growth factor binding-protein-3 (IGFBP

–

3). Best Pract Res Clin Endocrinol

Metab. 2015;29(5):701-711.

45- Serebryanaya DV, Adasheva DA, Konev AA, et al. IGFBP-4 proteol- ysis by PAPP-A in a primary

culture of rat neonatal cardiomyocytes under normal and hypertrophic conditions. Biochemistry

(Mosc). 2021;86(11):1395-1406.

46- Conover CA. Key questions and answers about pregnancy-associated plasma protein-A. Trends in

Endocrinology and Metabolism: TEM 2012;23:242

–

249.

47- Espelund US, Bjerre M, Hjortebjerg R, et al. 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. J Clin Endocrinol Metab. 2017;102(9):3526-3534.

ACCEPTED MANUSCRIPT

Downloaded from https://academic.oup.com/jes/advance-article/doi/10.1210/jendso/bvae081/7656891 by UCSD-Philosophy user on 23 April 2024

48- Holly JMP, Biernacka K, Perks CM. The neglected insulin: IGF-II, a metabolic regulator with

implications for diabetes, obesity, and cancer. Cells.

2019;8(10):1207.

49- Barrios V, Martín-Rivada Á, Guerra-Cantera S, et al. Reduction in Pappalysin-2 levels and Lower

IGF-I Bioavailability in Female Adolescents With Anorexia Nervosa. J Clin Endocrinol Metab.

2024;109(3):920-931.

50- Barrios V, Martín Rivada Á, Martos-Moreno GÁ, et al. Increased IGFBP Proteolysis, IGF-I

Bioavailability, and Pappalysin Levels in Children With Prader-Willi Syndrome. J Clin Endocrinol

Metab. 2023;

https://doi.org/10.1210/clinem/dgad754

51- Jones JI, Clemmons DR. Insulin-like growth factors and their binding proteins: biological actions.

Endocr Rev. 1995;16 (1):3-34.

52- Clemmons DR. Metabolic actions of insulin-like growth factor-I in normal physiology and

diabetes. Endocrinol Metab Clin N Am. 2012;41:425

–

443.

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FIGURE LEGENDS

Figure 1. Flowchart of data collection.

Tick (v) represents timepoints when samples/measurements were taken. Cross (x):

represents timepoints without such information. N/A: Not available or not applicable.

Figure 2. Boxplot representation of serum concentrations of total and free or intact forms

of IGFs and IGFBPs, pappalysins, stanniocalcins and ALS, at times T0, T1 and T2.

A: STC2 SDS; B: PAPP-A2 SDS; C: Total IGF-I SDS; D: Free IGF-I SDS; E: Total IGFBP-3 SDS; F: Intact

IGFBP-3 SDS; G: Total IGFBP-4 SDS; H: Intact IGFBP-4 SDS. I: IGF-II SDS; J: IGFBP-5 SDS; K: ALS SDS; L:

IGFBP-2 SDS; M: STC1 SDS; N: PAPP-A SDS. *p<0.05, ** p<0.01, ***p<0.001

Figure 3. Schematic cartoon of the biochemical findings in the peripheral GH -IGF axis in children

with T1DM

From T0, to T1 and T2 there is progressive increase in the concentrations of STC2, paralleled by

decrease in PAPP-A2 and overall increase in most forms of total IGFs and IGFBPs. There is significant

increase in intact IGFBP-3, but non-

significant for other free forms. “Arrows” indicate the direction of

change, if found; “equal” symbol indicates the lack of relevant change in concentrations of analyte

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Table 1. Research Resource Identifier (RRID) of the assays used.

Parameters

Commercial source

Catalog #

RRID

Total IGF-I

Ansh Labs

AL-121

AB_2783672

Free IGF-I

Ansh Labs

AL-122

AB_2783673

IGF-II

Ansh Labs

AL-131

AB_2783680

IGFBP-2

Ansh Labs

AL-140

AB_2783686

Total IGFBP-3

Ansh Labs

AL-120

AB_2783671

Intact IGFBP-3

Ansh Labs

AL-149

AB_2783688

Total IGFBP-4

Ansh Labs

AL-126

AB_2783676

Intact IGFBP-4

Ansh Labs

AL-128

AB_2783678

IGFBP-5

Ansh Labs

AL-127

AB_2783677

ALS

Mediagnost

E35

AB_2813809

Insulin

BioVendor

RIS006R

AB_2893123

PAPP-A

Ansh Labs

AL-101

AB_2783656

PAPP-A2

Cloud Clone

SCD471Hu

AB_2893124

STC-1

R&D Systems

DY2958

AB_2893122

STC-2

Ansh Labs

AL-143

AB_2783687

Abbreviations: ALS, acid-labile subunit; IGF, insulin-like growth factor; IGFBP-2, insulin-like growth

factor binding protein-2; IGFBP-3, insulin-like growth factor binding protein-3; IGFBP-4, insulin-like

growth factor binding protein-4; IGFBP-5, insulin-like growth factor binding protein-5; PAPP-A,

pregnancy-associated plasma protein A; PAPP-A2, pregnancy-associated plasma protein A2; STC-1,

stanniocalcin 1; STC-2, stanniocalcin 2.

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Table 2. Comparison of HbA1c, height SD and BMI SD at T0, T1 and T2.

Variable

T1 versus T0

T2 versus T0

Adjusted

Hb1Ac

-3.99 CI95% (-4.94,-3.03)
P<0.001

-3.66 CI95% (-4.81,-2.05)
P<0.001

0.0001***

0.5187

Height SD

-0.23 CI95% (-0.74,0.26)
P=0.350

-0.18 CI95% (-0.79,0.43)
P=0.565

0.6113

-0.0129

BMI SD

0.13 CI95% (-0.46,0.71)
P=0.668

0.30 CI95% (-0.42,1.02)
P=0.411

0.6962

-0.0164

BMI: Body mass index; CI: confidence interval; HbA1c: glycated hemoglobin; m: months; SD:

standard deviation; ***p<0.001

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Table 3. Comparison of growth velocity and nutritional markers between T1 and T2.

Variable

T2 versus T1

Adjusted

GV SD

0.23 CI95% (-0.72, 1.19)

0.6245

-0.0234

Prealbumin

1.82 CI95% (-0.37,4.00)

0.1001

0.0660

Transferrin

30.91 CI95% (0.36,61.47)

0.0476*

0.1348

Retinol

-0.00 CI95% (-0,20,0.20)

0.9769

-0.0384

GV: growth velocity; SD: standard deviation; *p<0.05

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Table 4. Serum concentrations of pappalysins, stanniocalcins, ALS, total and intact IGF

and IGFBPs.

ANOVA p value of T0 versus T1 (*p<0.05, ** p<0.01, ***p<0.001) and T0 versus T2 (#p<0.05, ##

p<0.01, ###p<0.001). IQR: Interquartile range; SD: standard deviation.

Analyte (SD)

T0 [median (IQR)]

T1 [median (IQR)]

T2 [median (IQR)]

Total IGF-I

-1.20 (-1.56,-0.50)

-0.04 (-0.77,0.3)***

-0.53 (-0.89,0.25)

Free IGF-I

-1.59 (-1.78,-1.18)

-1.56 (-1.8-0.97)

-1.29 (-1.8,0.65)

Total IGF-II

0.54 (0.01,1.68)

1.08 (0.39,1.63)

1.71 (1.08,2.41)

ALS

-0.94 (-1.59, 0.15)

0.53 (-0.24,1.18)***

0.41 (0.11,1.01)

IGFBP-2

-0.48 (-1.42,0.49)

-0.83 (-1.43,-0.07)

-0.47 (-1.19,0.12)

Total IGFBP-3

0.15 (-0.71,0.88)

0.24 (-0.32,0.44)

0.34 (-0.13,0.73)

Intact IGFBP-3

-1.07 (-1.89,-0.62)

-0.22 (-0.97,0.11)***

-0.09 (-1.01,0.15)

Total IGFBP-4

-0.24 (-0.64,0.24)

-0.16 (-0.58,0.97)

0.98 (0.45,1.32)

###

Intact IGFBP-4

-0.72 (-1.04,-0.23)

-0.47(-0.73,0.45)

-0.13 (-0.47,0.65)

IGFBP-5

-0.93 (-1.35,-0.03)

0.45 (-1.8,-0.97)***

1.95 (0.72,2.42)

###

STC1

-1.23 (-1.56,-0.84)

-1.19 (-1.37,-0.94)

-1.26 (-1.35,-0.85)

STC2

-1.31 (-1.97,-0.39)

0.07 (-0.37,0.69)***

0.52 (-0.76,0.90)

###

PAPP-A

0.63 (-0.08,1.33)

0.80 (-0.3,1.27)

0.93 (0.76,1.92)

PAPPA-A2

1.05 (0.68,1.63)

0.49 (-0.07,0.92)***

0.13 (-0.16,0.52)

###

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Table 5. Correlations.

Significant correlations (P<0.05) were found between the analyte in first

column and those in the second column.

HbA1c

PAPPA-2 (r: 0.41), STC2 (r: -0.32), intact IGFBP-3 (r: -0.29), transferrin (r: 0.45),
prealbumin (r: -0.38)

PAPP-A

STC1 (r: -0.27), free IGF-I (r: 0.35), total IGF-II (r: 0.29)

STC1

ALS (r: -0.28), total IGF-2 (r: -0.23), intact IGFBP-4 (r: 0.26)

STC2

STC1 (r: -0.23), ALS (r: 0.37), total IGF-2 (r: 0.34), prealbumin (r: 0.64)

Free IGF-I

STC2 (r: 0.29), ALS (r: 0.25), total IGF-II (r: 0.53), IGFBP-2 (r: -0.49), intact IGFBP-4
(r: -0.40), total IGFBP-4 (r: 0.37), prealbumin (r: 0.56)

Intact IGFBP-3

STC2 (r: 0.39), ALS (r: 0.63), total IGF-II (r: 0.34), prealbumin (r: 0.51), weight SD
(r: 0.22)

ALS

Total IGF-II (r: 0.33), IGFBP-2 (r: -0.34)

IGFBP-2

Total IGF-II (r: -0.39), prealbumin (r: -0.40)

Intact IGFBP-4

IGFBP-2 (r: 0.34), prealbumin (r: -0.51), retinol binding protein (r: -0.47)

Total IGF-II

Total IGFBP-4 (r: 0.26), BMI SD (r: 0.22), weight SD (r: 0.25)

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