Increased Proteolysis of Insulin-Like Growth Factor-Binding Protein-3 (IGFBP-3) in Noninsulin-Dependent Diabetes Mellitus Serum, with Elevation of a 29-Kilodalton (kDa) Glycosylated IGFBP-3 Fragment Contained in the Approximately 130- To 150-kDa Ternary Complex

Peter Bang, Kerstin Brismar, Ronald (Ron) Rosenfeld

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Abstract

Insulin-like growth factor-I (IGF-I) in serum is predominantly bound in a ternary complex, consisting of IGF peptide, IGF-binding protein-3 (IGFBP-3), and an acid-labile subunit, or a binary complex, consisting of IGF peptide and any of the six IGFBPs. In the binary complex, IGF-I is more bioavailable and has a faster turnover rate. Proteolysis of IGFBP-3 may alter the distribution of IGF-I between these complexes by reducing IGFBP-3 affinity for IGF-I and/or acid-labile subunit and may offer an additional mechanism for regulation of IGF availability. In the present study, sera from patients with noninsulin-dependent diabetes mellitus (NIDDM) were found to have significantly higher IGFBP-3 proteolytic activity than sera from age-matched healthy subjects (188 ± 12% vs. 104 ± 6% of a control serum pool; P <0.001). The mean (±SE) of serum IGFBP-3 levels determined by Western ligand blotting was lower in NIDDM patients than in healthy control subjects (61.5 ± 5% and 79 ± 5% of a control serum pool, respectively; P <0.01). However, IGFBP-3 concentrations determined by RIA did not differ. This discrepancy could be explained by IGFBP-3 proteolysis, resulting in IGFBP-3 fragments that are detectable by RIA, but not by Western ligand blotting. Western immunoblotting of sera with or without prior treatment with endoglycosidase-F demonstrated that a glycosylated 29-kilodalton (kDa) IGFBP-3 form with a protein core of 20 kDa was present in sera from healthy controls, and this fragment was increased in NIDDM and term pregnancy sera, suggesting that it is produced by endogenous proteolysis. The presence of the 29-kDa IGFBP-3 proteolytic fragment at about 130-150 kDa after neutral size chromatography of pooled sera may suggest that 29-kDa IGFBP-3 participates in ternary complex formation. Further studies are required to determine whether the avidity of ternary complex formation with the 29-kDa IGFBP-3 fragment is reduced and whether the resulting increased IGF turnover can explain the reduced IGF-I levels (z scores) observed in NIDDM patients compared to healthy subjects (-0.81 ± 0.32 SD vs. +0.26 ± 0.17 SD; P <0.001). Neutral size-chromatography of sera demonstrated that IGFBP-3 protease activity in the approximately 130- to 150-kDa mol wt range is regulated by NIDDM and pregnancy in parallel with that of unfractionated sera. In addition, all sera tested, including sera from healthy subjects, demonstrated high IGFBP-3 proteolytic activity in the approximately 40- to 50-kDa mol wt range. It may be hypothesized that lack of an inhibitor of the approximately 40- to 50-kDa protease(s), which is normally present, may contribute to increased IGFBP-3 protease activity in NIDDM and term pregnancy sera. The increased IGFBP-3 proteolytic activity in NIDDM serum was partially cation dependent and inhibited by serine protease inhibitors, similar to that of term pregnancy sera.

Original languageEnglish (US)
Pages (from-to)1119-1127
Number of pages9
JournalJournal of Clinical Endocrinology and Metabolism
Volume78
Issue number5
StatePublished - May 1994
Externally publishedYes

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Proteolysis
Insulin-Like Growth Factor Binding Protein 3
Medical problems
Type 2 Diabetes Mellitus
Serum
Insulin-Like Growth Factor I
Healthy Volunteers
Pregnancy
Chromatography
glycosylated insulin
Western Blotting
Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase
Ligands
Insulin-Like Growth Factor Binding Proteins
Peptides
Serine Proteinase Inhibitors
Acids

ASJC Scopus subject areas

  • Biochemistry
  • Endocrinology, Diabetes and Metabolism

Cite this

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title = "Increased Proteolysis of Insulin-Like Growth Factor-Binding Protein-3 (IGFBP-3) in Noninsulin-Dependent Diabetes Mellitus Serum, with Elevation of a 29-Kilodalton (kDa) Glycosylated IGFBP-3 Fragment Contained in the Approximately 130- To 150-kDa Ternary Complex",
abstract = "Insulin-like growth factor-I (IGF-I) in serum is predominantly bound in a ternary complex, consisting of IGF peptide, IGF-binding protein-3 (IGFBP-3), and an acid-labile subunit, or a binary complex, consisting of IGF peptide and any of the six IGFBPs. In the binary complex, IGF-I is more bioavailable and has a faster turnover rate. Proteolysis of IGFBP-3 may alter the distribution of IGF-I between these complexes by reducing IGFBP-3 affinity for IGF-I and/or acid-labile subunit and may offer an additional mechanism for regulation of IGF availability. In the present study, sera from patients with noninsulin-dependent diabetes mellitus (NIDDM) were found to have significantly higher IGFBP-3 proteolytic activity than sera from age-matched healthy subjects (188 ± 12{\%} vs. 104 ± 6{\%} of a control serum pool; P <0.001). The mean (±SE) of serum IGFBP-3 levels determined by Western ligand blotting was lower in NIDDM patients than in healthy control subjects (61.5 ± 5{\%} and 79 ± 5{\%} of a control serum pool, respectively; P <0.01). However, IGFBP-3 concentrations determined by RIA did not differ. This discrepancy could be explained by IGFBP-3 proteolysis, resulting in IGFBP-3 fragments that are detectable by RIA, but not by Western ligand blotting. Western immunoblotting of sera with or without prior treatment with endoglycosidase-F demonstrated that a glycosylated 29-kilodalton (kDa) IGFBP-3 form with a protein core of 20 kDa was present in sera from healthy controls, and this fragment was increased in NIDDM and term pregnancy sera, suggesting that it is produced by endogenous proteolysis. The presence of the 29-kDa IGFBP-3 proteolytic fragment at about 130-150 kDa after neutral size chromatography of pooled sera may suggest that 29-kDa IGFBP-3 participates in ternary complex formation. Further studies are required to determine whether the avidity of ternary complex formation with the 29-kDa IGFBP-3 fragment is reduced and whether the resulting increased IGF turnover can explain the reduced IGF-I levels (z scores) observed in NIDDM patients compared to healthy subjects (-0.81 ± 0.32 SD vs. +0.26 ± 0.17 SD; P <0.001). Neutral size-chromatography of sera demonstrated that IGFBP-3 protease activity in the approximately 130- to 150-kDa mol wt range is regulated by NIDDM and pregnancy in parallel with that of unfractionated sera. In addition, all sera tested, including sera from healthy subjects, demonstrated high IGFBP-3 proteolytic activity in the approximately 40- to 50-kDa mol wt range. It may be hypothesized that lack of an inhibitor of the approximately 40- to 50-kDa protease(s), which is normally present, may contribute to increased IGFBP-3 protease activity in NIDDM and term pregnancy sera. The increased IGFBP-3 proteolytic activity in NIDDM serum was partially cation dependent and inhibited by serine protease inhibitors, similar to that of term pregnancy sera.",
author = "Peter Bang and Kerstin Brismar and Rosenfeld, {Ronald (Ron)}",
year = "1994",
month = "5",
language = "English (US)",
volume = "78",
pages = "1119--1127",
journal = "Journal of Clinical Endocrinology and Metabolism",
issn = "0021-972X",
publisher = "The Endocrine Society",
number = "5",

}

TY - JOUR

T1 - Increased Proteolysis of Insulin-Like Growth Factor-Binding Protein-3 (IGFBP-3) in Noninsulin-Dependent Diabetes Mellitus Serum, with Elevation of a 29-Kilodalton (kDa) Glycosylated IGFBP-3 Fragment Contained in the Approximately 130- To 150-kDa Ternary Complex

AU - Bang, Peter

AU - Brismar, Kerstin

AU - Rosenfeld, Ronald (Ron)

PY - 1994/5

Y1 - 1994/5

N2 - Insulin-like growth factor-I (IGF-I) in serum is predominantly bound in a ternary complex, consisting of IGF peptide, IGF-binding protein-3 (IGFBP-3), and an acid-labile subunit, or a binary complex, consisting of IGF peptide and any of the six IGFBPs. In the binary complex, IGF-I is more bioavailable and has a faster turnover rate. Proteolysis of IGFBP-3 may alter the distribution of IGF-I between these complexes by reducing IGFBP-3 affinity for IGF-I and/or acid-labile subunit and may offer an additional mechanism for regulation of IGF availability. In the present study, sera from patients with noninsulin-dependent diabetes mellitus (NIDDM) were found to have significantly higher IGFBP-3 proteolytic activity than sera from age-matched healthy subjects (188 ± 12% vs. 104 ± 6% of a control serum pool; P <0.001). The mean (±SE) of serum IGFBP-3 levels determined by Western ligand blotting was lower in NIDDM patients than in healthy control subjects (61.5 ± 5% and 79 ± 5% of a control serum pool, respectively; P <0.01). However, IGFBP-3 concentrations determined by RIA did not differ. This discrepancy could be explained by IGFBP-3 proteolysis, resulting in IGFBP-3 fragments that are detectable by RIA, but not by Western ligand blotting. Western immunoblotting of sera with or without prior treatment with endoglycosidase-F demonstrated that a glycosylated 29-kilodalton (kDa) IGFBP-3 form with a protein core of 20 kDa was present in sera from healthy controls, and this fragment was increased in NIDDM and term pregnancy sera, suggesting that it is produced by endogenous proteolysis. The presence of the 29-kDa IGFBP-3 proteolytic fragment at about 130-150 kDa after neutral size chromatography of pooled sera may suggest that 29-kDa IGFBP-3 participates in ternary complex formation. Further studies are required to determine whether the avidity of ternary complex formation with the 29-kDa IGFBP-3 fragment is reduced and whether the resulting increased IGF turnover can explain the reduced IGF-I levels (z scores) observed in NIDDM patients compared to healthy subjects (-0.81 ± 0.32 SD vs. +0.26 ± 0.17 SD; P <0.001). Neutral size-chromatography of sera demonstrated that IGFBP-3 protease activity in the approximately 130- to 150-kDa mol wt range is regulated by NIDDM and pregnancy in parallel with that of unfractionated sera. In addition, all sera tested, including sera from healthy subjects, demonstrated high IGFBP-3 proteolytic activity in the approximately 40- to 50-kDa mol wt range. It may be hypothesized that lack of an inhibitor of the approximately 40- to 50-kDa protease(s), which is normally present, may contribute to increased IGFBP-3 protease activity in NIDDM and term pregnancy sera. The increased IGFBP-3 proteolytic activity in NIDDM serum was partially cation dependent and inhibited by serine protease inhibitors, similar to that of term pregnancy sera.

AB - Insulin-like growth factor-I (IGF-I) in serum is predominantly bound in a ternary complex, consisting of IGF peptide, IGF-binding protein-3 (IGFBP-3), and an acid-labile subunit, or a binary complex, consisting of IGF peptide and any of the six IGFBPs. In the binary complex, IGF-I is more bioavailable and has a faster turnover rate. Proteolysis of IGFBP-3 may alter the distribution of IGF-I between these complexes by reducing IGFBP-3 affinity for IGF-I and/or acid-labile subunit and may offer an additional mechanism for regulation of IGF availability. In the present study, sera from patients with noninsulin-dependent diabetes mellitus (NIDDM) were found to have significantly higher IGFBP-3 proteolytic activity than sera from age-matched healthy subjects (188 ± 12% vs. 104 ± 6% of a control serum pool; P <0.001). The mean (±SE) of serum IGFBP-3 levels determined by Western ligand blotting was lower in NIDDM patients than in healthy control subjects (61.5 ± 5% and 79 ± 5% of a control serum pool, respectively; P <0.01). However, IGFBP-3 concentrations determined by RIA did not differ. This discrepancy could be explained by IGFBP-3 proteolysis, resulting in IGFBP-3 fragments that are detectable by RIA, but not by Western ligand blotting. Western immunoblotting of sera with or without prior treatment with endoglycosidase-F demonstrated that a glycosylated 29-kilodalton (kDa) IGFBP-3 form with a protein core of 20 kDa was present in sera from healthy controls, and this fragment was increased in NIDDM and term pregnancy sera, suggesting that it is produced by endogenous proteolysis. The presence of the 29-kDa IGFBP-3 proteolytic fragment at about 130-150 kDa after neutral size chromatography of pooled sera may suggest that 29-kDa IGFBP-3 participates in ternary complex formation. Further studies are required to determine whether the avidity of ternary complex formation with the 29-kDa IGFBP-3 fragment is reduced and whether the resulting increased IGF turnover can explain the reduced IGF-I levels (z scores) observed in NIDDM patients compared to healthy subjects (-0.81 ± 0.32 SD vs. +0.26 ± 0.17 SD; P <0.001). Neutral size-chromatography of sera demonstrated that IGFBP-3 protease activity in the approximately 130- to 150-kDa mol wt range is regulated by NIDDM and pregnancy in parallel with that of unfractionated sera. In addition, all sera tested, including sera from healthy subjects, demonstrated high IGFBP-3 proteolytic activity in the approximately 40- to 50-kDa mol wt range. It may be hypothesized that lack of an inhibitor of the approximately 40- to 50-kDa protease(s), which is normally present, may contribute to increased IGFBP-3 protease activity in NIDDM and term pregnancy sera. The increased IGFBP-3 proteolytic activity in NIDDM serum was partially cation dependent and inhibited by serine protease inhibitors, similar to that of term pregnancy sera.

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