Association of the insulin-like growth factor binding protein 3 (IGFBP-3) polymorphism with longevity in Chinese nonagenarians and centenarians

Human lifespan is determined greatly by genetic factors and some investigations have identified putative genes implicated in human longevity. Although some genetic loci have been associated with longevity, most of them are difficult to replicate due to ethnic differences. In this study, we analyzed the association of 18 reported gene single nucleotide polymorphisms (SNPs) with longevity in 1075 samples consisting of 567 nonagenarians/centenarians and 508 younger controls using the GenomeLab SNPstream Genotyping System. Our results confirm the association of the forkhead box O3 (FOXO3) variant (rs13217795) and the ATM serine/threonine kinase (ATM) variant (rs189037) genotypes with longevity (p=0.0075 and p=0.026, using the codominant model and recessive model, respectively). Of note is that we first revealed the association of insulin-like growth factor binding protein 3 (IGFBP-3) gene polymorphism rs11977526 with longevity in Chinese nonagenarians/centenarians (p=0.033 using the dominant model and p=0.035 using the overdominant model). The FOXO3 and IGFBP-3 form important parts of the insulin/insulin-like growth factor-1 signaling pathway (IGF-1) implicated in human longevity, and the ATM gene is involved in sensing DNA damage and reducing oxidative stress, therefore our results highlight the important roles of insulin pathway and oxidative stress in the longevity in the Chinese population.     

Spleen stromal cells support haemopoiesis and in vitro growth of dendritic cells from bone marrow

Long-term stroma-dependent cultures from murine spleen have been previously shown to support dendritic cell (DC) development in vitro. Secondary cultures have now been established using a splenic stromal cell layer overlaid with cells from different lymphoid sites. Cells resembling DCs can be generated in vitro from unfractionated murine lymphoid cells in the absence of added growth factors. Bone marrow (BM) cultures are the most successful but some cultures have been derived from spleen and thymus. Large numbers of mononuclear cells with dendritic morphology can be generated from BM within 2 weeks and cell production in many cultures has been maintained for at least 6 months. A significant proportion of cells binds antibodies specific for DC markers. No lymphoid cells, mast cells or granulocytes are detectable in culture by antibody and histochemical staining and light and electron microscopy. As with cells generated in primary cultures, cells grown in secondary cultures are equally potent stimulators of both syngeneic and allogeneic mixed lymphocyte reactions, confirming their function as antigen-presenting cells. They are also capable of endocytosing and presenting protein antigen to the D10.G4.1 Th2 clone and to unprimed T cells. This study confirmed the presence of DC precursors in multiple lymphoid sites which can be induced to proliferate in the presence of a spleen stromal cell monolayer. The secondary culture system provides an ideal in vitro model for investigation of the development of DC from different tissue sites. It also provides a stable and continuous resource of cells for further studies on DC development.     

Circulating salmon 41-kDa insulin-like growth factor binding protein (IGFBP) is not IGFBP-3 but an IGFBP-2 subtype

In vertebrates, most circulating insulin-like growth factor (IGF) is bound to multiple forms of IGF-binding proteins (IGFBPs) that differ both structurally and functionally. In mammals, the largest reservoir of IGF in the circulation comes from a large (150 kDa) ternary complex comprised of IGF bound to IGFBP-3, which is bound to an acid label subunit (ALS), and this variant of IGFBP is regulated by growth hormone (GH) and feed intake. Although multiple variants of IGFBPs ranging from 20 to 50 kDa have been found in fishes, no ternary complex is present and it has been assumed that the majority of circulating IGF is bound to fish IGFBP-3. Consistent with this assumption is previous work in salmon showing the presence of a 41-kDa IGFBP that is stimulated by GH, decreases with fasting and increases with feeding. However, the hypothesis that the salmon 41-kDa IGFBP is structurally homologous to mammalian IGFBP-3 has not been directly tested. To address this issue, we cloned cDNAs for several Chinook salmon IGFBPs, and found that the cDNA sequence of the 41-kDa IGFBP is most similar to that of mammalian IGFBP-2 and dissimilar to IGFBP-3. We found an additional IGFBP (termed IGFBP-2a) with high homology to mammalian IGFBP-2. These results demonstrate that salmon 41-kDa IGFBP is not IGFBP-3, but a paralog of IGFBP-2 (termed IGFBP-2b). Salmon IGFBP-2s are also unique in terms of having potential N-glycosylation sites and splice variants. Additional research on non-mammalian IGFBPs is needed to fully understand the molecular/functional evolution of the IGFBP family and the significance of the ternary complex in vertebrates.     

Characterization of TGF-β1-binding proteins in human bone marrow stromal cells

The proliferation and differentiation of haemopoietic progenitor cells is dependent on their close relation with bone marrow stromal cells, which constitute a source of cytokines as well as expressing receptors for both the cytokines and progenitor cell adhesion molecules necessary for regulated haemopoiesis. We have generated human bone marrow stromal cell cultures and analysed the TGF-β1 receptor components expressed by these cells. [¹²⁵I]TGF-β1-affinity labelling experiments showed the involvement of type I and II receptors in the binding of TGF-β1, as demonstrated by specific immunoprecipitation of [¹²⁵I]TGF-β1–receptor complexes. In addition, large TGF-β1-labelled complexes displaying an electrophoretic mobility similar to betaglycan were also observed in these experiments. Endoglin, another component of the TGF-βreceptor system, was detected by flow cytometry on the surface of cultured marrow stromal cells, and in the human bone marrow stromal cell line Str-5, and was immunoprecipitated from surface-iodinated cells. Endoglin on the stromal cells was able to bind TGF-β1, as demonstrated by specific immunoprecipitation of [¹²⁵I]TGF-β1–endoglin complexes using anti-endoglin antibodies. The results presented provide evidence that bone marrow stromal cells are fully capable of responding to TGF-β1. Given the important role of TGF-βas a regulator of the synthesis of cytokines and cytokine receptors, as well as cell adhesion molecules, these data indicate that the binding of TGF-β1 by stromal cells might represent an important step in the regulation of the proliferation and differentiation of haemopoietic progenitor cells.     

Circulating salmon 28- and 22-kDa insulin-like growth factor binding proteins (IGFBPs) are co-orthologs of IGFBP-1

Circulating insulin-like growth factor binding proteins (IGFBPs) play pivotal roles in stabilizing IGFs and regulating their availability to target tissues. In the teleost circulation, three major IGFBPs are typically detected by ligand blotting with molecular masses around 20-25, 28-32 and 40-45 kDa. However, their identity is poorly established and often confused. We previously identified salmon 22- and 41-kDa forms as IGFBP-1 and -2b, respectively. In the present study, we cloned the cDNA of 28-kDa IGFBP from Chinook salmon (Oncorhynchus tshawytscha) as well as rainbow trout (Oncorhynchus mykiss) based on the partial N-terminal amino acid sequence of purified protein and identified it as an ortholog of IGFBP-1. Structural and phylogenetic analyses revealed that the 28-kDa IGFBP is more closely related to human IGFBP-1 and zebrafish IGFBP-1a than the previously identified salmon IGFBP-1 (i.e. 22-kDa IGFBP). We thus named salmon 28- and 22-kDa forms as IGFBP-1a and -1b, respectively. Salmon IGFBP-1a contains a potential PEST region involved in rapid protein turnover and phosphorylation sites typically found in mammalian IGFBP-1, although the PEST and phosphorylation scores are not as high as those of human IGFBP-1. There was a striking difference in tissue distribution patterns between subtypes; Salmon igfbp-1a was expressed in a variety of tissues while igfbp-1b was almost exclusively expressed in the liver, suggesting that IGFBP-1a has more local actions. Direct seawater exposure (osmotic stress) of Chinook salmon parr caused increases in both IGFBP-1s in plasma, while IGFBP-1b appeared to be more sensitive. The presence of two co-orthologs of IGFBP-1 in the circulation in salmon, and most likely in other teleosts, provides a good opportunity to investigate subfunction partitioning of duplicated IGFBP-1 during postnatal growth.     

白血病干细胞和骨髓基质细胞共培养的方法学探讨

目的 探讨白血病干细胞和骨髓基质细胞共培养的可行方法及扩增白血病干细胞的特点.方法 体外用两种方法共培养急性髓系白血病（AML）患者的骨髓单个核细胞（MNCs）：一种方法是将MNCs中的贴壁基质细胞单独培养,第2代或第3代基质细胞用丝裂霉素处理后做饲养层,再同悬浮造血细胞共培养;另一种方法是将MNCs持续培养即原代共培养.观察MNCs形态学特征、长期存活情况及增殖特性,用流式细胞仪检测分析细胞表面标志.结果 两种方法共培养的细胞部分可以长期存活,在共培养中有卵石样区域细胞及悬浮细胞团的形成.原代共培养的细胞所形成的卵石样区域数目多,排列规律.结论 两种方法都可以起到扩增白血病干细胞的作用.同经过丝裂霉素处理之后的饲养层相比,没有经过处理的骨髓基质细胞在共培养时能够更有效地扩增白血病干细胞.     

Regulation of insulin-like growth factor binding protein-1 (IGFBP-1) in insulin-dependent diabetes mellitus

The aim of the present study was to characterize the effect of 44 h of hyperglycaemia on diurnal levels of insulin-like growth factor binding protein-1 (IGFBP-1), insulin-like growth factor-1 (IGF-1), growth hormone (GH) and glucagon in 7 well-controlled subjects with insulin-dependent diabetes mellitus (IDDM). Hyperglycaemia (15 mmol/l) was induced by a glucose infusion, while the degree of insulinisation was similar to that of a corresponding period with near normoglycaemia (6.9 mmol/l). Hyperglycaemia for 44 h did not alter the normal diurnal IGFBP-1 levels when the degree of insulinisation was unchanged. The diurnal secretion pattern of IGFBP-1 was preserved in both genders and without any difference between the control and hyperglycaemic periods. However, the IGFBP-1 levels were increased in these IDDM subjects despite a peripheral hyperinsulinemia. An inverse correlation was found between IGFBP-1 and peripheral insulin levels both during periods of rapid changes in IGFBP-1 and insulin concentrations (i.e. morning hours) as well as during the total 24-h sampling period. Total IGF-1 levels were low, but no further decrease was seen after 24 h of hyperglycaemia in the presence of unchanged insulin levels. In conclusion, the present study clearly shows that the increased IGFBP-1 level seen during poor metabolic control in IDDM is not caused by hyperglycaemia. Glucose levels per se do not influence either total IGF-1 or IGFBP-1 concentrations in well-insulinised diabetic patients.     

Studies on regulation of insulin-like growth factor binding protein (IGFBP)-3 and IGFBP-4 production in human bone cells.

Previous studies have shown that the actions of IGF-II in bone are determined not only by its concentration, but also by the concentration of IGFBP-4 as well as other IGFBPs. In this study, we sought to determine by Western ligand blotting the effects of growth hormone, IGF-I and IGF-II on the production of IGFBP-3 and IGFBP-4 in TE89 human osteosarcoma cells and in untransformed normal human bone cells derived from rib. Human growth hormone at 10 micrograms/l decreased the amount of IGFBP-4 but had no effect on the IGFBP-3 level in the conditioned medium of low density cultures of TE89 cells and human bone cells derived from rib. Human growth hormone had no effect on IGFBP-3 or IGFBP-4 levels in the conditioned medium of high density human bone cell cultures. IGF-I and IGF-II, which increased human bone cell proliferation, decreased the level of IGFBP-4 (30% of control at 100 micrograms/l IGF-I and IGF-II) but increased the level of IGFBP-3 (3-10 fold at 100 micrograms/l IGF-I and IGF-II) after 48 h of treatment in the conditioned medium of both low and high density TE89 cell cultures. Similar changes in IGFBP-3 and IGFBP-4 levels were also seen in the conditioned medium of human bone cells derived from rib after treatment with IGF-I and IGF-II. Studies to determine the underlying molecular mechanisms by which IGF-II decreased the amount of IGFBP-4 in the conditioned medium revealed that IGF-II decreased the IGFBP-4 mRNA abundance and increased the IGFBP-3 mRNA abundance in human bone cells. Based on the above findings, we conclude that the production of both IGFBP-3 and IGFBP-4 is regulated in bone cells and that local and systemic agents may modulate the responsiveness of bone cells to IGFs by regulated secretion of IGFBP-3 and IGFBP-4.     

老年住院病人血清胰岛素样生长因子-1、胰岛素样生长因子结合蛋白-3与衰弱的相关性研究

目的探讨老年住院病人血清胰岛素样生长因子-1(IGF-1)、胰岛素样生长因子结合蛋白-3(IGFBP-3)水平与衰弱的相关性。方法纳入2018年1月至2019年6月老年医学科年龄≥65岁的住院病人195例,进行衰弱表型评估和老年综合评估,采用ELISA法检测血清IGF-1、IGFBP-3水平,分析其与衰弱的相关性。结果与无衰弱、衰弱前期病人相比,衰弱病人血清IGF-1、IGFBP-3水平显著下降,差异有统计学意义(P<0.05)。有序多分类Logistic回归分析显示血清IGF-1(OR=0.943,95%CI0.894~0.994,P<0.05)、IGFBP-3(OR=0.397,95%CI0.259~0.607,P<0.001)水平与衰弱显著相关。结论血清IGF-1、IGFBP-3水平与老年住院病人衰弱显著相关,可能成为老年人衰弱的预测指标。     

Interaction between insulin-like growth factor binding protein-related protein 1 and trans-forming growth factor beta 1 in primary hepatic stellate cells

BACKGROUND:We previously showed that insulin-like growth factor binding protein-related protein 1(IGFBPrP1) is a novel mediator in liver fibrosis.Transforming growth factor beta 1(TGFβ1) is known as the strongest effector of liver fibrosis.Therefore,we aimed to investigate the detailed interaction between IGFBPrP1 and TGFβ1 in primary hepatic stellate cells(HSCs).METHODS:We overexpressed TGFβ1 or IGFBPrP1 and inhibited TGFβ1 expression in primary HSCs for 6,12,24,48,72,and 96 hours to investigate their interaction and observe the accompanying expressions of α-smooth muscle actin(α-SMA),collagen I,fibronectin,and phosphorylated-mothers against decapentaplegic homolog 2/3(p-Smad2/3).RESULTS:We found that the adenovirus vector encoding the TGFβ1 gene(Ad TGFβ1) induced IGFBPrP1 expression while that of α-SMA,collagen I,fibronectin,and TGFβ1 increased gradually.Concomitantly,Ad IGFBPrP1 upregulated TGFβ1,α-SMA,collagen I,fibronectin,and p-Smad2/3 in a time-dependent manner while IGFBPrP1 expression was decreased at 96 hours.Inhibition of TGFβ1 expression reduced the IGFBPrP1-stimulated expression of α-SMA,collagen I,fibronectin,and p-Smad2/3.CONCLUSIONS:These findings for the first time suggest the existence of a possible mutually regulation between IGFBPrP1 and TGFβ1,which likely accelerates liver fibrosis progression.Furthermore,IGFBPrP1 likely participates in liver fibrosis in a TGFβ1-depedent manner,and may act as an upstream regulatory factor of TGFβ1 in the Smad pathway.     

大鼠骨髓基质细胞培养方法的改良

目的　对体外分离培养大鼠骨髓基质细胞 (BMSC)方法进行改良 ,观察其生物学特点。方法　分离大鼠胫骨、股骨 ,以 IMDM培养基冲洗骨髓 ,与培养液混合后直接接种至培养瓶中 ,接种后 7～ 14 d形成单层贴壁的成纤维状细胞。检测传代细胞接种贴壁率、生长曲线、细胞周期 ,电镜观察细胞超微结构。结果　分离培养的原代 BMSC生长良好 ,倍增时间约为 4 2 h。 BMSC传代后 12 h贴壁率达 80 %以上 ,82 %的细胞处于 G0 、G1 期 ,超微结构呈现较早期细胞特点。结论　改良法培养的 BMSC生长稳定 ,传代细胞适应性强。与传统培养方法比 ,该法操作简单、实用性强 ,可以用于 BMSC的体外实验研究     

Expression regulation of insulin-like growth factor binding proteins (IGFBP). Tissue-specific expression of IGFBP-1]

We have investigated the liver-specific expression of IGFBP-1 gene, an Insulin-like Growth Factor binding protein. Northern blot as well as transient transfection experiments, both carried out in differentiated (H4II, C2Rev7) and dedifferentiated (H5, C2) hépatoma cell lines, yielded clear cut data allowing the involvement of HNF1, a liver-specific trans acting factor, in basal IGFBP-1 liver-specific expression.     

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

Summary The 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.     

Effect of intraperitoneal insulin delivery on growth hormone binding protein,insulin-like growth factor (IGF)-I,and IGF-binding protein-3 in IDDM

Summary Low plasma insulin-like growth factor (IGF)-I despite high circulating growth hormone (GH) in insulin-dependent diabetes mellitus (IDDM) indicate a hepatic GH resistance. This state may be reflected by the reduction of the circulating GH binding protein (GHBP), corresponding to the extracellular domain of the GH receptor, and the reduction of insulin-like growth factor binding protein (IGFBP)-3, major IGF-I binding protein, upregulated by GH. We carried out two studies. In the first, plasma GHBP activity was compared in patients with IDDM on continuous subcutaneous insulin infusion (CSII) or on conventional therapy and in healthy subjects. In the second study, the 18 patients on CSII at baseline were then treated by continuous intraperitoneal insulin infusion with an implantable pump (CPII) and prospectively studied for GH-IGF-I axis. Although HbA_{1 c} was lower in patients on CSII than in those on conventional therapy, GHBP was similarly reduced in both when compared to control subjects (10.2 ± 0.8 and 11.6 ± 0.9 % vs 21.0 ± 1.3, p < 0.01). CPII for 12 months resulted in: a slight and transient improvement in HbA_{1 c} (Time (T)0: 7.6 ± 0.2 %, T3:7.1 ± 0.2 %, T12: 7.5 ± 0.2 %, p < 0.02), improvement in GHBP (T0: 10.2 ± 0.8 %, T12: 15.5 ± 1.5, p < 0.0001), near-normalization of IGF-I (T0: 89.4 ± 8.8 ng/ml, T12: 146.9 ± 15.6, p < 0.002) and normalization of IGFBP-3 (T0: 1974 ± 121 ng/ml, T12: 3534 ± 305, p < 0.0001). The hepatic GH resistance profile in IDDM does not seem to be related to glycaemic control, but partly to insufficient portal insulinization. Intraperitoneal insulin delivery, allowing primary portal venous absorption, may influence GH sensitivity, and improve hepatic IGF-I and IGFBP-3 generation. [Diabetologia (1996) 39: 1498–1504] Received: 23 March 1996 and in revised form: 22 July 1996     

Characterization of bone marrow mesenchymal stromal cells in aplastic anaemia

In aplastic anaemia (AA), haemopoietic activity is significantly reduced and generally attributed to failure of haemopoietic stem cells (HSC) within the bone marrow (BM). The regulation of haemopoiesis depends on the interaction between HSC and various cells of the BM microenvironment, including mesenchymal stromal cells (MSC). MSC involvement in the functional restriction of HSC in AA is largely unknown and therefore, the physical and functional properties of AA MSC were studied in vitro. MSC were characterized by their phenotype and ability to form adherent stromal layers. The functional properties of AA MSC were assessed through proliferative, clonogenic and cross‐over culture assays. Results indicate that although AA MSC presented typical morphology and distinctive mesenchymal markers, stromal formation was reduced, with 50% of BM samples failing to produce adherent layers. Furthermore, their proliferative and clonogenic capacity was markedly decreased (P = 0·03 and P = 0·04 respectively) and the ability to sustain haemopoiesis was significantly reduced, as assessed by total cell proliferation (P = 0·032 and P = 0·019 at Week 5 and 6, respectively) and clonogenic potential of HSC (P = 0·02 at Week 6). It was concluded that the biological characteristics of AA MSC are different from those of control MSC and their in vitro haemopoiesis‐supporting ability is significantly reduced.     

Insulin-like growth factor binding protein (IGFBP) substrate zymography

Insulin-like growth factor binding protein (IGFBP) degrading proteinase activities have been described in biological fluids and conditioned media from numerous cell lines. To identify and characterize IGFBP-degrading proteinases, our laboratory has developed IGFBP substrate zymography. Herein, we illustrate how IGFBP substrate zymography can be used both to identify candidate IGFBP-degrading proteinases and characterize their degradative capabilities. For this purpose, human matrix metalloproteinase-3 (MMP-3), a proteinase that degrades IGFBP-3 in human fibroblast cultures, was first electrophoresed through a polyacrylamide gel containing IGFBP-3 as substrate and then analyzed for its ability to degrade the substrate into immunoreactive fragments that were abssorbed onto a polyvinylidene difluoride membrane. IGFBP-3 substrate zymography was capable of detections as little as 20 ng of human MMP-3, demonstrating a sensitivity similar to casein substrate zymography. Using the zymogram as a template, MMP-3 was identified in a standard SDS-polyacrylamide gel run in parallel with the zymogram, and the corresponding area of the gel was excised. Electroelution of the gel slice yielded active MMP-3 when examined by casein substrate zymography. Furthermore, digestion of IGFBP-3 in solution by the electroeluted MMP-3 revealed the same fragmentation pattern of the binding protein as that produced by MMP-3, which had not been electroeluted. Together, these studies demonstrate that IGFBP substrate zymography can be a useful tool for both the identification and the characterization of IGFBP-degrading proteinases.     

Decreased production of insulin-like growth factor-binding protein (IGFBP)-6 by transfection of colon cancer cells with an antisense IGFBP-6 cDNA construct leads to stimulation of cell proliferation

BACKGROUND: : Previously, we have observed that highly unsaturated dietary (n-3) fatty acids inhibit cell proliferation in conjunction with stimulation of insulin-like growth factor-binding protein (IGFBP)-6 secretion in Caco-2 cells, a human colon carcinoma cell line. METHODS:: To test the converse hypothesis that inhibition of endogenous IGFBP-6 secretion stimulates Caco-2 cell proliferation, cells were transfected with the antisense IGFBP-6 expression construct or pcDNA3 vector only, and single colonies resistant to G418 sulfate were isolated. RESULTS:: Our initial studies indicated that three antisense clones grew faster and produced less IGFBP-6 than two pcDNA3 clones, so antisense IGFBP-6 #5 and pcDNA3 #8 were selected for further detailed analysis. Both the control and antisense clones grew in serum-free medium reaching a plateau density at day eight. However, the antisense clone grew at a rate faster than that of the control and reached a final density that was 31 +/- 3% higher than the control. Northern blot, ligand blot and immunoblot analyses revealed that accumulation of IGFBP-6 mRNA and concentrations of IGFBP-6 peptide produced by the antisense clone were decreased by 80-90% compared to the control. The doubling times of the antisense and control clones were 21.9 +/- 0.4 and 24.8 +/- 0.3 h (P < 0.05), respectively. Exogenous IGF-I and IGF-II (0.2-200 nmol/L) stimulated proliferation of both the control and antisense clones in a dose-dependent manner, but the relative potency and efficacy of IGF-II was higher in the antisense clone compared to the control. These results indicate that suppression of IGFBP-6 secretion correlates with an increase in the basal rate of Caco-2 cell growth. CONCLUSIONS:: Our findings are consistent with the hypothesis that IGFBP-6 inhibits cell growth by binding to endogenously produced IGF-II, thereby preventing IGF-II from interacting with the IGF-I receptor to stimulate cellular proliferation by an autocrine mechanism.     

Metabolic actions of insulin-like growth factor II in cultured adult rat hepatocytes are not mediated through the insulin-like growth factor II receptor

Summary Short- and long-term regulation of hepatic carbohydrate metabolism by insulin-like growth factor II was studied in primary cultures of adult rat hepatocytes and compared to the metabolic potency of insulin. Insulin-like growth factor II stimulated glycogen synthesis from [¹⁴C]glucose, uptake of [³H]aminoisobutyric acid and [¹⁴C]lactate formation from [¹⁴C]glucose up to three-fold. Basal glycogenolysis was inhibited to about 10%, and glucagon-activated glycogenolysis was blocked completely. The enzymatic activity of glucokinase and pyruvate kinase was induced two-fold, the glucagon-dependent induction of phosphoenolpyruvate carboxykinase was antagonized. Compared to insulin, half-maximal responses required up to 50 times higher insulin-like growth factor II concentrations ranging from 10–20 nmol/l. A similar difference was observed for binding affinity of insulin-like growth factor II to the insulin receptor. The interaction with the insulin-like growth factor II/man-nose 6-phosphate (IGF-II/Man-6-P) receptor was examined by studying ¹²⁵I-insulin-like growth factor II binding and uptake of lysosomal enzymes. The affinity of insulin-like growth factor II to the IGF-II/Man-6-P receptor was considerably higher than for the insulin receptor. Antibodies against the IGF-II/Man-6-P receptor did not affect metabolic responses to insulin-like growth factor II, while binding to its receptor and the receptor-mediated endocytosis of arylsulphatase A were strongly inhibited. Thus, in adult rat liver insulin-like growth factor II appeared to exert metabolic actions not via interaction with its own receptor but through low affinity binding to hepatic insulin receptors.     

Insulin-like growth factor binding proteins in the bovine anterior pituitary

Insulin-like growth factor binding proteins (IGFBPs) were characterized in bovine anterior pituitary tissue, pituitary conditioned media, and serum collected during the preovulatory and early luteal phases of the estrous cycle. Effects of in vitro treatments of pituitaries with luteinizing hormone-releasing hormone (LHRH), estradiol, and progesterone on IGFBP secretion were also evaluated. Predominant IGFBPs detected in anterior pituitary tissue by immunoprecipitation, ligand blotting, and Northern blotting were IGFBP-5 (29 kDa), IGFBP-2 (32 kDa), and IGFBP-3 (36 and 39 kDa doublet). Conditioned culture media contained IGFBP-5, a slightly larger form of IGFBP-3 (33 kDa), the 36- and 39-kDa forms of IGFBP-3, and a more extensively glycosylated form of IGFBP-3 (44 kDa). In serum, IGFBP-5 was not readily detected, and IGFBP-3 (40- and 44-kDa doublet) and IGFBP-2 (34 kDa) were larger than in pituitary tissue. Levels of IGFBP-2,-3, and-5 in pituitary tissue decreased during the preovulatory period and were lowest in the early luteal phase. Treatment with LHRH increased IGFBP-2 levels in media twofold. Estradiol or progesterone did not alter IGFBP secretion in vitro. Predominant IGFBPs produced and released by anterior pituitary tissue were IGFBP-2,-3 and-5. The activity of IGFBPs fluctuates in the pituitary in association with changes in stage of estrous cycle, implicating IGFBPs as potential regulators of gonadotrope function. Mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture.