Wieland Kiess

Transient increase in renal insulin-like growth factor binding proteins during initial kidney hypertrophy in experimental diabetes in rats

SummaryThe insulin-like growth factors, insulin-like growth factor I and insulin-like growth factor II are bound to six distinct classes of insulin-like growth factor binding proteins (IGFBPs) in the circulation and in extracellular fluids. Diabetic renal hypertrophy is preceded by a transient increase in kidney insulin-like growth factor I suggestive of a renotropic function for insulin-like growth factor I. In order to examine a possible involvement of IGFBPs in initial diabetic kidney growth and in kidney insulin-like growth factor I accumulation, we studied rat kidney IGFBPs by ligand blotting during the first 4 days after induction of diabetes. Six distinct bands were identified in kidney and liver tissue with apparent molecular weight values of 38–47 (doublet), 34, 30, 24 and 20 kDa. The 38–47 kDa doublet band probably corresponds to the insulin-like growth factor binding subunit of IGFBP-3, the 24 kDa band to IGFBP4 and the 30 kDa band to IGFBP-1 and/or IGFBP-2, as these IGFBPs in rats have similar molecular weight. In untreated diabetic rats a transient increase in the kidney 30 kDa band was demonstrable 24 h after induction of diabetes with a maximal rise (two-fold) after 48 h, followed by a decrease to baseline values after 4 days. In untreated diabetic rats the 38–47 kDa doublet band also increased (two-fold) in kidney during the first 2 days after induction of diabetes, followed by a subsequent decrease. Insulin-treatment prevented both the increase in the 30 kDa and in the 38–47 kDa bands. Kidney weight in untreated diabetic rats increased by 26 % after 4 days. In conclusion, the present study shows a transient increase in the 30 kDa and the 38–47 kDa IGFBP species in hypertrophying diabetic kidneys, contemporarily with the previously described transient increase in extractable kidney insulin-like growth factor I content. These findings support the concept that IGFBPs may be involved in the action of insulin-like growth factor I and possibly in the diabetic kidney insulin-like growth factor I accumulation.

Insulin-Like Growth Factor II (IGF-II) and the IGF-II/ Mannose-6-Phosphate Receptor: The Myth Continues

Insulin-like growth factor II (IGF-II) is a polypeptide hormone with structural and functional homology with IGF-I and pro-insulin. It is now thought that IGF-II acts as a growth factor during fetal life and development. In rats, IGF-II levels in the circulation are high in the fetus and decline rapidly after birth. IGF-II mRNA expression in many tissues including the liver and the choroid plexus is also high during fetal life and low thereafter. Targeted disruption of the IGF-II gene in mice leads to a deficiency in their growth. Alternatively, it has been proposed that IGF-II could act as a growth and differentiation factor in the central nervous system. Administration of IGF-II into the central nervous system in rats leads to increased food intake and altered feeding behaviour. In muscle cells and in colon epithelial cells, IGF-II might also serve as an important regulator of differentiation. A key role for IGF-II as a paracrine or autocrine growth factor in certain tumours has been proposed. The IGFs exert their effects by binding to high-affinity membrane receptors that are expressed in many cells and tissues. The IGF-I receptor, which binds IGF-I with the highest affinity and which is very similar to the insulin receptor, is thought to mediate most, if not all, of the IGF-induced biological functions. The IGF-II/mannose-6-phosphate (M6P) receptor is a bifunctional glycoprotein with no homology to the insulin receptor. This receptor binds IGF-II and lysosomal enzymes bearing the M6P recognition marker at distinct binding sites.(ABSTRACT TRUNCATED AT 250 WORDS)

Reciprocal Modulation of Binding of Lysosomal Enzymes and Insulin-Like Growth Factor-II (IGF-II) to the Mannose 6- Phosphate/IGF-II Receptor

The discovery by Morgan et al.1 that the human IGF-II receptor is 80% homologous to the bovine cation-independent mannose 6-phosphate (Man-6-P) receptor led them to propose that the receptor is bifunctional, binding both a large number of lysosomal enzymes through the Man-6-P recognition site, and the growth factor, IGF-II. Later, Komfeld’s and Czech’s laboratories reported that the avian and amphibian Man-6-P receptors did not bind IGF-II, suggesting that the bifunctional property of the receptor may be confined to mammals.2,3 Did the mammalian receptor gene simply pick up a nucleotide sequence encoding an IGF-II binding site, enabling the receptor to provide a degradative pathway for IGF-II, or are there important interactions of the two disparate classes of ligands for binding to the receptor? These interactions could result in reciprocal modulation of the targeting of lysosomal enzymes by IGF-II, on the one hand, and the modulation of IGF-II degradation and IGF-II stimulated biologic responses by lysosomal enzymes, on the other. We will briefly summarize our experimental results which provide evidence for reciprocal inhibition of binding of the two classes of ligands for the mammalian Man-6-P/ IGF-II receptor.

Human colon carcinoma cells (CaCo-2) synthesize IGF-II and express IGF-I receptors and IGF-II/M6P receptors.

The IGFs have been implicated in the development of the intestinal tract. We have studied the human colon carcinoma cell line CaCo-2 to gain more insight into the function of the IGFs in the gut. [125I]IGF-I and -II bound specifically to CaCo-2 cells as measured in competitive binding experiments. The existence of IGF-I receptors was further demonstrated by affinity crosslinking studies using DSS as the crosslinking agent. Western blotting of CaCo-2 cell extracts using an anti IGF-II/M6P receptor antiserum provided additional evidence for the expression of the IGF-II/M6P receptor. In addition, Northern blotting experiments showed specific IGF-I receptor and IGF-II/M6P receptor gene expression in CaCo-2 cells. An 11 kb band was visualized with a 614 bp PstI IGF-I receptor probe on autoradiographs. Hybridization with a 663 bp IGF-II/M6P receptor probe yielded a 9 kb RNA species. Analysis of CaCo-2 cell RNA using solution hybridization/RNase protection assays yielded two protected fragments, approximately 379 bases in length, with a 394 base IGF-I receptor riboprobe and a 250 base protected fragment with a 260 base IGF-II/M6P receptor riboprobe. In a subset of experiments a PstI 700 base fragment of the IGF-I cDNA and a 554 base SalI fragment of the IGF-II cDNA were used for hybridization: no hybridization was detected with the IGF-I probe. However, using the [32P]IGF-II probe bands at 6.0 and 5.0 kb were labeled in Northern blotting experiments. Analysis of CaCo-2 cell RNA using solution hybridization/RNase protection assays yielded a 289 base protected fragment and a faint 534 base species with a 556 base human IGF-II riboprobe. In addition, IGF-II immunoreactivity was measured in CaCo-2 cell-conditioned medium using an IGF-binding protein blocked radioimmunoassay. CaCo-2 cell-conditioned medium contained 5-15 ng/ml IGF-II immunoreactivity. In conclusion, (1) CaCo-2 cells express both IGF-I receptor mRNA and IGF-II/M6P receptor mRNA and contain functional IGF-I receptor and IGF-II/M6P receptor protein. (2) CaCo-2 cells express IGF-II mRNA and secrete IGF-II immunoreactivity. We hypothesize that in human colon carcinoma cells IGF-II could act as an autocrine growth factor or alternatively could serve as a regulatory factor during differentiation.

The Insulin-Like Growth Factor-II/Mannose-6-Phosphate Receptor: Structure, Function and Differential Expression

The insulin like growth factor-II/mannose-6-phosphate (IGF-II/M6P) receptor is a bifunc-tional binding protein that binds lysosomal enzymes bearing the M6P recognition marker and IGF-II at distinct binding sites (45, 52). In addition, transforming growth factor (TGF) beta precursor, thyroglobulin and proliferin, a protein which is expressed in rapidly proliferating cells are also recognized by this receptor (Table 1). In avian and amphibian cells the receptor lacks the binding site for IGF-II but serves as a binding protein for M6P bearing ligands (7,9,76). Almost all mammalian cells described until today express IGF-II/M6P receptors that bind both classes of ligands, namely M6P-containing glycoproteins and IGF-II (55–59).

Human IM-9 lymphoblasts as a model of the growth hormone-insulin-like growth factor axis: gene expression, and interactions of ligands with receptors and binding proteins

Human IM-9 lymphoblasts bind growth hormone (hGH) and insulin-like growth factors (IGFs). We have systematically examined the IM-9 cells as a valuable model of the interaction of hGH and the IGFs at the cellular level. Cells were cultured in medium with 10% serum and for a subset of experiments cultured in serum-free medium. Binding of [125I]hGH and [125I]IGF-I and -II to intact IM-9 cells was measured: unlabeled hGH inhibited binding of [125I]hGH (half max. 20 ng/ml). Binding of [125I]IGF-I was inhibited by IGF-I (half max. 7.5 ng/ml), IGF-II (half max. 60 ng/ml), and insulin and anti IGF-I receptor antibody (alpha IR3). [125I]IGF-II was inhibited by IGF-II (half max. 15 ng/ml), IGF-I (half max. 500 ng/ml), insulin (half max. 250 ng/ml) but not by alpha IR3. Crosslinking experiments with [125I]IGF-II and DSS as the crosslinking agent and analysis of radioligand-receptor complexes by SDS-PAGE under reducing conditions revealed that [125I]IGF-II bound to a 250 kDa and a 135 kDa receptor species. The latter possibly represents an insulin-type receptor whereas the 250 kDa species had the characteristics of the IGF-II/M6P receptor. When IM-9 cell conditioned medium was analyzed in ligand blotting experiments with either [125I]IGF-I or -II a 30 kDa IGFBP species was detected on the autoradiographs. Also, IGF-II immunoreactivity (approx. 1 ng/ml medium) was measured in the cell conditioned medium using an IGF-BP blocked RIA employing [125I]IGF-II. In a subset of experiments IM-9 cells were homogenized in 4 M guanidinium-thiocyanate and RNA extracted in 5.7 M CsCl. Denatured RNA was electrophoresed on 0.8% agarose gels and transferred to a nylon membrane, fixed and the blots hybridized with cDNA probes. Probes were labeled with [32P]dCTP using a random prime labeling procedure: a Pst I 700 bp fragment of the human IGF-I cDNA, a 554 bp Pst I-Sal I fragment of the IGF-II cDNA, a 614 bp Pst I fragment of the IGF-I receptor cDNA and a 663 bp Pst I fragment of the IGF-II/M6P receptor. Autoradiographs of Northern blots showed specific hybridization with the IGF-I probe at 3.7 kb and with the IGF-II probe at 5.3 kb. No signal was detected with the IGF-I receptor cDNA probe. Hybridization with the IGF-II/M6P receptor probe yielded a 9 kb RNA species.(ABSTRACT TRUNCATED AT 400 WORDS)

Measurement of Spontaneous, 12-Hour Sleep-Associated GH Secretion in Prepubertal Children with Short Stature: Clinical Relevance and Practicability?

We have asked whether or not sleep-associated 12-hour GH profiles were a clinically relevant and practicable tool to identify short children with low spontaneous GH secretion. In 67 prepubertal patients (19 girls and 48 boys, mean age 9.34 years, range 1.99-14.5) sleep-associated 12-hour GH profiles were obtained by drawing peripheral venous blood every 30 min over a 12-hour night period. The diagnosis of GH deficiency (GHD, n = 26), constitutional delay of puberty and growth (CDPG, n = 19), familial short stature (FSS, n = 8), GH neurosecretory dysfunction (GHND, n = 5), and constitutional delay of puberty and growth plus familial short stature (CDPGFSS, n = 9) was made by clinical parameters (SDS height range:-0.69 to -5.59, SDS growth velocity:-4.6 to -2.4) and provocative testing of GH secretion. Integrated GH secretion (area above baseline = AOB, area above zero line = AOOL), peak frequency, area under the peaks, peak amplitude length, peak amplitude height, maximal peak values, and median peak values were calculated using the PULSAR program. Significant differences of GH secretion between patient groups in regard to mean values for area over baseline, area over zero line, amplitude height, maximal peak values, and median peak values of secretion were found. However, there was a large interindividual variation of integrated GH secretion within each patient group and, most importantly, a large overlap between the different patient groups. We conclude that the assessment of pulsatile GH secretion during sleep, even if it can contribute to distinguish between different groups of short children, is not helpful to distinguish between different causes of short stature in an individual child. We suggest that measurement of sleep-associated spontaneous GH secretion needs to be restricted to research facilities.

Pertussis toxin sensitive G-proteins are not involved in the mitogenic signaling pathway of insulin-like growth factor-I in normal rat kidney epithelial (NRKE) cells

There is controversy as to whether or not a pertussis toxin sensitive G-protein is involved in the signaling pathway of insulin-like growth factor-I. We have used normal rat kidney epithelial (NRKE) cells to ask whether or not a pertussis toxin sensitive G-protein was involved in IGF-I stimulated DNA synthesis. NRKE cells express both IGF and IGF-II/M6P receptors and respond to IGF-I with increased thymidine incorporation into DNA. Under many circumstances incubation of cells/cell membranes with GTP analogues will inhibit binding of ligands that are linked to a G-protein-receptor pathway. However, when NRKE membrane preparations were incubated with 125I-IGF-I or 125I-IGF-II in the presence or absence of GTP gamma S, ATP and GTP, binding of the radioligands was not affected by the GTP-analogue. IGF-I and factors from serum of hypophysectomized rats (HRS) stimulated [3H]thymidine incorporation into DNA of NRKE cells. Under serum-free conditions in the presence of EGF (2 ng/ml) and PDGF (1 ng/ml) pertussis toxin over a wide range of doses had no effect upon IGF-I stimulated [3H]thymidine incorporation into DNA of NRKE cells. In addition, PT at a dose of 100 ng/ml had no effect on IGF-I(0.2-50 ng/ml) stimulated DNA synthesis of NRKE cells. However, PT at doses of 5, 50, 500, 5000 and 50,000 ng/ml was capable to ADP-ribosylate a 40 kDa protein in NRKE cell plasma membrane preparations corresponding to known PT-sensitive G-proteins. We conclude, that (1) PT-sensitive G-proteins and both IGF-I and IGF-II/M6P receptors are present in NRKE cell plasma membrane preparations, and most importantly, that (2) PT-sensitive G-proteins are not involved in the mitogenic signaling pathway of IGF-I in NRKE cells.

The insulin-like growth factor-II/mannose-6-phosphate receptor is present in fetal bovine tissues throughout gestation

The insulin-like growth factor-II (IGF-II) and the IGF-II/mannose-6-phosphate (M6P) receptor are thought to play an important role in fetal growth and development. We have studied the expression of the IGF-II/M6P receptor in fetal bovine tissues from 5 through 36 weeks gestation. Tissues from bovine fetuses were extracted in buffer containing 2% Triton-X-100 and 2% sodium dodecyl sulfate (SDS). Aliquots of the protein extracts were analyzed by SDS polyacrylamide gel electrophoresis and the protein bands were transferred onto nitrocellulose. Immunoblotting was performed with anti-bovine IGF-II/M6P receptor antiserum. In a subset of experiments, ligand blotting was carried out with radiolabeled IGF-II and subsequent autoradiography. IGF-II/M6P receptors were expressed in all tissues examined, with the highest amount of receptor being present in fetal lung and liver. Low amounts of receptors were measured in fetal brain. The amount of receptor was developmentally regulated throughout fetal life. The developmental regulation of receptor expression varied among the different tissues. In conclusion, the IGF-II/M6P receptor is present in all fetal bovine tissues examined. The presence of the IGF-II/M6P receptor seems to be developmentally regulated during bovine fetal life. We hypothesize that this receptor exerts important biologic effects during fetal growth and tissue and organ development.

Evidence against a role for insulin-like growth factor II in the autonomous growth of rat 18,54-SF cells

The 18,54-SF rat cell line multiplies in serum-free medium and has been reported to produce insulin-like growth factor II (IGF-II) and to possess IGF-II/mannose 6-phosphate (Man-6-P) receptors, raising the possibility of autocrine growth stimulation by IGF-II acting through this receptor. When serum-free medium was changed every 24 h the 18,54-SF cells multiplied at the same rate as when the medium was not changed. An antibody (No. 3637) which blocks the binding of IGF-II to the IGF-II/Man-6-P receptor did not decrease or increase the growth rate of the 18,54-SF cells when medium was changed every 24 h. When the medium was changed every 12 h the rate of accumulation of cells in the culture was decreased. Addition of IGF-I or IGF-II at 10, 50, and 100 ng/ml every 12 h did not correct this decrease in cell number. When the medium was replaced every 12 h, cells in the periphery of the cultures gradually became nonviable as assessed by trypan blue uptake. However, the percentage of cells synthesizing DNA in the center of the cultures (approximately 50% during a 3 h pulse with tritiated thymidine) was the same whether the medium was changed every 12 h or left unchanged. Addition of IGF-I or IGF-II to the fresh medium change every 12 h did not increase the percentage of cells synthesizing DNA.