Tumor necrosis factor-alpha-induced sickness behavior is impaired by central administration of an inhibitor of c-jun N-terminal kinase.

RATIONALE: Tumor necrosis factor-alpha (TNFalpha) acts within the brain to induce sickness behavior, but the molecular mechanisms are still unknown. TNFalpha binding induces receptor trimerization, activation of c-Jun N-terminal kinase (JNK), and induction of downstream transcription factors. OBJECTIVES: We hypothesized that TNFalpha-induced sickness behavior can be blocked by a novel JNK inhibitor. METHODS: To test this idea, we used a bipartite protein consisting of a ten-amino-acid sequence of the trans-activating domain of the viral TAT protein (D-TAT) linked to a 19-amino-acid peptide that specifically inhibits JNK activation (D-JNKI-1). C57BL/6J mice were pre-treated intracerebroventricularly (i.c.v.) with D-JNKI-1 or the control peptide containing only the protein transduction domain, D-TAT. Mice were then injected centrally with an optimal amount of TNFalpha (50 ng/mouse) to induce sickness behavior. Sickness was assessed as a decrease in social exploration of a novel juvenile, an increase in duration of immobility and loss of body weight. RESULTS: Pre-treatment with D-JNKI-1 (10 ng/mouse), but not D-TAT, significantly inhibited all three indices of sickness induced by central TNFalpha. CONCLUSIONS: These findings demonstrate that D-JNKI-1 can abrogate TNFalpha-induced sickness behavior and suggest a potential therapeutic target for treating major depressive disorders that develop on a background of cytokine-induced sickness behavior.

Prion biology relevant to bovine spongiform encephalopathy.

Bovine spongiform encephalopathy (BSE) and chronic wasting disease (CWD) of deer and elk are a threat to agriculture and natural resources, as well as a human health concern. Both diseases are transmissible spongiform encephalopathies (TSE), or prion diseases, caused by autocatalytic conversion of endogenously encoded prion protein (PrP) to an abnormal, neurotoxic conformation designated PrPsc. Most mammalian species are susceptible to TSE, which, despite a range of species-linked names, is caused by a single highly conserved protein, with no apparent normal function. In the simplest sense, TSE transmission can occur because PrPsc is resistant to both endogenous and environmental proteinases, although many details remain unclear. Questions about the transmission of TSE are central to practical issues such as livestock testing, access to international livestock markets, and wildlife management strategies, as well as intangible issues such as consumer confidence in the safety of the meat supply. The majority of BSE cases seem to have been transmitted by feed containing meat and bone meal from infected animals. In the United Kingdom, there was a dramatic decrease in BSE cases after neural tissue and, later, all ruminant tissues were banned from ruminant feed. However, probably because of heightened awareness and widespread testing, there is growing evidence that new variants of BSE are arising "spontaneously," suggesting ongoing surveillance will continue to find infected animals. Interspecies transmission is inefficient and depends on exposure, sequence homology, TSE donor strain, genetic polymorphism of the host, and architecture of the visceral nerves if exposure is by an oral route. Considering the low probability of interspecies transmission, the low efficiency of oral transmission, and the low prion levels in nonnervous tissues, consumption of conventional animal products represents minimal risk. However, detection of rare events is challenging, and TSE literature is characterized by subsequently unsupported claims of species barriers or absolute tissue safety. This review presents an overview of TSE and summarizes recent research on pathogenesis and transmission.

Zinc partitions IGFs from soluble IGF binding proteins (IGFBP)-5, but not soluble IGFBP-4, to myoblast IGF type 1 receptors.

Zinc (Zn(2+)), a multifunctional micronutrient, was recently shown to lower the affinity of cell-associated insulin-like growth factor (IGF) binding protein (IGFBP)-3 and IGFBP-5 for both IGF-I and IGF-II, but to increase the affinity of the cell surface type 1 IGF receptor (IGF-1R) for the same two ligands. However, there is a need for data concerning the effects of Zn(2+) on soluble IGFBPs and the type 2 IGF receptor (IGF-2R). In the current work, we demonstrate that Zn(2+) affects the affinity of IGFBP-5 secreted by myoblasts but not IGFBP-4. Zn(2+), at physiological levels, depressed binding of both IGF-I and IGF-II to IGFBP-5, affecting (125)I-IGF-I more than (125)I-IGF-II. Both (125)I-IGF-I and (125)I-IGF-II bound to high and low affinity sites on IGFBP-5. Zn(2+) converted the high affinity binding sites of IGFBP-5 into low affinity binding sites. An IGF-I analog, (125)I-R(3)-IGF-I, did not bind to the soluble murine IGFBP-5. Zn(2+) also decreased the affinity of the IGF-2R on L6 myoblasts. In contrast, Zn(2+) increased IGF-I, IGF-II and R(3)-IGF-I binding to the IGF-1R by increasing ligand binding affinity on both P(2)A(2a)-LISN and L6 myoblasts. Soluble IGFBP-5 and IGFBP-4 depressed the binding of (125)I-IGF-I and (125)I-IGF-II to the IGF-1R, but did not affect binding of (125)I-R(3)-IGF-I. By depressing the association of the IGFs with soluble IGFBP-5, Zn(2+) partitioned (125)I-IGF-I and (125)I-IGF-II from soluble IGFBP-5 onto cell surface IGF-1Rs. This effect is not seen when soluble L6-derived IGFBP-4 is present in extracellular fluids. We introduce a novel mechanism by which the trace micronutrient Zn(2+) may alter IGF distribution, i.e. Zn(2+) acts to increase IGF-1R binding at the expense of IGF binding to soluble IGFBP-5 and the IGF-2R.

Controlling insulin-like growth factor activity and the modulation of insulin-like growth factor binding protein and receptor binding.

The insulin-like growth factors (IGF) and insulin perform seemingly unique roles by causing the same metabolic effect: cellular hypertrophy. Although overlapping, there are different consequences to cellular hypertrophy induced by IGF and that induced by insulin. The IGF enhance the cell hypertrophy that is requisite for cell survival, hyperplasia, and differentiation, and insulin enhances cell hypertrophy primarily as a means to increase nutrient stores. The effects of IGF and insulin are controlled by the segregation of their receptors between different cell types. A model is discussed that describes the need for three hormones (IGF-I, IGF-II, and insulin) to control nutrient partitioning. Insulin receptor localization, as well as an episodic mode of secretion, evolved to perform the short-term action of clearing excess nutrients from the circulation. In contrast, a complex and interactive set of factors ensure that maximal IGF activity occurs only when conditions are optimal for growth. A relatively invariant rate of secretion and the IGF binding proteins serve to maintain a large mutable pool of IGF. This pool exists to ensure a constant supply of IGF to maintain the basal metabolic rate and to ensure that, once a cell begins to proliferate or differentiate, adequate exposure is available to complete the process even after severe short-term physiological insults. The IGF concentrations only change in response to prolonged differences in protein and energy availabilities, environmental and body temperatures, and external stress. Also, evidence is now emerging that describes a discrete role for trace nutrients in the regulation of IGF activity. In this latter regard, zinc has the notable role of targeting IGF binding proteins to the cell surface. New data are presented showing that zinc also changes the affinity of the type 1 IGF receptor and cell-associated IGF binding proteins to optimize IGF activity.

Multivalent cations and ligand affinity of the type 1 insulin-like growth factor receptor on P2A2-LISN muscle cells.

Mouse P2A2-LISN myoblasts are transfected cells that overexpress the human type 1 insulin-like growth factor (IGF) receptor. Because the type 1 IGF receptor is the major binding site for both IGF-I and IGF-II, this cell line is an excellent model to determine the effect of multivalent cations on ligand binding specifically to this type of receptor. Competitive binding assays were performed to characterize IGF binding and Scatchard analysis to quantify affinity (Ka). 125I-IGF-I, 125I-IGF-II, and 125I-R3-IGF-I bind only to the type 1 IGF receptor on these cells. Zn2+ increased binding of the three ligands to the type 1 IGF receptor by 17 to 35%. Cd2+ significantly increased binding of 125I-IGF-I, although by only 8%. La3+ and Cr3+ did not effect binding. Au3+ decreased IGF binding by approximately 56%. Scatchard analysis produced nonlinear concave-down plots yielding binding constants for high and low affinity sites. Zn2+ increased the strength of only the high affinity sites. Au3+ decreased the affinity of both high and low affinity sites. Zn2+ increased binding with a half-maximal effect between 40 microM and 60 microM. Half-maximal dose of Au3+ was >130 microM. Zinc, gold, and cadmium bind to similar regions within proteins (a zinc-binding motif) and only these cations were found to affect receptor binding indicating similar mechanisms of action. Thus, multivalent cations may alter IGF binding to cell surface receptors ultimately controlling growth. Physiologically this may be especially important for the growth promoting effects of Zn2+.

Multivalent cations depress ligand affinity of insulin-like growth factor-binding proteins-3 and -5 on human GM-10 fibroblast cell surfaces.

The effect of multivalent cations on [125I]-IGF binding to cell-associated IGFBPs was investigated using human fibroblasts. The major cell-associated binding site for [125I]-IGF-I is IGFBP-3 and for [125I]-IGF-II are IGFBP-3 and IGFBP-5. Lanthanum and chromium did not affect either [125I]-IGF-I or [125I]-IGF-II binding to cell-associated IGFBPs. By contrast, zinc (Zn2+), gold (Au3+), and cadmium (Cd2+) depressed binding of both ligands. Ligand binding resulted in nonlinear Scatchard plots. Assuming a pre-existent asymmetric model with high- (K[aHi]) and low- (K[aLo]) affinity sites, Zn2+ lowered both K(aHi) and K(aLo). Au3+ eliminated K(aHi). Assuming that the nonlinear plots were caused by ligand-induced negative cooperativity, Zn2+ and Cd2+ lowered both Ke and Kf (affinity of unoccupied and saturated IGFBPs, respectively). Au3+ eliminated Ke and reduced Kf. Zn2+ was active at serum levels in lowering IGF binding. Zinc, gold, and cadmium bind to similar regions within proteins (a zinc-binding motif) indicating similar mechanisms of action. A zinc-binding motif is present in the IGFBPs, but not in the IGFs. We demonstrate for the first time that the trace nutrient zinc and related multivalent cations decrease IGF binding to fibroblast-associated IGFBPs by lowering the affinity of the IGF-IGFBP interaction.

Production of insulin-like growth factor I (IGF-I) and IGF-binding proteins by rat intestinal stromal cells in vitro.

To determine if intestinal stromal cells secrete diffusible factors such as insulin-like growth factors (IGFs) capable of regulating epithelial cell growth in vitro, stromal cells were isolated by enzymatic digestion of rat intestine. Incorporation of [3H]thymidine into DNA and [14C]leucine into protein of IEC-6 cells, a model intestinal epithelial cell line, was significantly increased (two- to threefold) when the IEC-6 cells were co-cultured with stromal cells, relative to IEC-6 cells grown alone. Medium conditioned by stromal cells stimulated DNA synthesis of IEC-6 cells in a dose-dependent manner. Analysis of the conditioned medium revealed that intestinal stromal cells secreted IGF-I, but little IGF-II, in addition to an M(r) 32,000 IGF-binding protein (IGFBP-2) and an IGFBP having M(r) approximately 24,000. We conclude that rat intestinal stromal cells secrete one or more diffusible factors, which may include IGF-I and IGFBPs, capable of stimulating proliferation of IEC-6 cells in vitro.

Multivalent cations depress ligand binding to cell-associated insulin-like growth factor binding protein-5 on human glioblastoma cells.

The current studies quantified the effect of the multivalent cations zinc, cadmium, lanthanum, chromium, and gold (Zn2+, Cd2+, La3+, Cr3+, and Au3+) on [125I]-insulin-like growth factor ([125I]-IGF) binding to T98G human glioblastoma cells. The major binding site for the IGFs on T98G cells is IGF binding protein-5 (IGFBP-5), as determined by affinity labeling. Competitive binding studies, using either [125I]-IGF-I or [125I]-IGF-II, indicated that La3+ and Cr3+ did not affect [125I]-IGF-I or [125I]-IGF-II binding to cell-associated IGFBP-5. Zn2+, Au3+, and Cd2+ depressed binding of both [125I]-IGF-I and [125I]-IGF-II. [125I]-IGF-I and [125I]-IGF-II binding resulted in nonlinear concave-down Scatchard plots, indicating the presence of high- and low-affinity equilibrium constant of association (Ka) sites. Assuming a preexisting asymmetric model with independent high (KaHi) and low (KaLo) sites; Zn2+, Cd2+, and Au3+ eliminated KaHi and Zn2+, and Au3+ lowered KaLo, compared with control values. The same results were found, independent of whether [125I]-IGF-I or [125I]-IGF-II was used. Similarly, assuming a ligand-induced model of negative cooperativity, all three cations eliminated the initial affinity for the high affinity sites (Ka), whereas Zn2+ and Au2+ reduced the final affinity for the low affinity sites (Kf). Dose-response studies indicated that Zn2+, Au3+, and Cd2+ depressed binding with half-maximal activities of approximately 20 microM, 14-60 microM, and 50-65 microM, respectively. Zn2+, Au3+, and Cd2+ bind to similar sites on proteins (a zinc-binding motif), indicating similar mechanisms of action. A zinc-binding motif is present within the IGFBPs but not the IGFs. We demonstrate, for the first time, that multivalent cations have the potential to modulate IGF activity by decreasing the amount of IGF bound to cell-associated IGFBP-5.

Role for cyclic adenosine monophosphate in modulating insulin-like growth factor binding protein secretion by muscle cells.

The modulation of insulin-like growth factor-binding protein (IGFBP) secretion is an important variable affecting muscle cell metabolism, proliferation, and differentiation. We have previously shown that secretion of IGFBP-4 and IGFBP-5 by L6 and BC3H-1 muscle cells was stimulated by treatment with either insulin, IGF-I, or IGF-II. Herein, these cells were used to further identify mechanisms involved in controlling IGFBP secretion. Agents that elevate intracellular cAMP concentrations (dcAMP, forskolin, isoproterenol, and prostaglandin [PGE1]) increase secretion of IGFBP-4 and IGFBP-5 from L6 cells. Similar increases in IGFBP secretion were found by treatment with either insulin, IGF-I, or dcAMP. The effects of dcAMP and either insulin or IGF-I were additive, but the effects of insulin and IGF-I were not additive. These results suggest that insulin/IGF-I and dcAMP are acting via distinct mechanisms to stimulate IGFBP secretion. Indomethacin, which blocks endogenous prostaglandin synthesis, and progesterone, which decreases intracellular cAMP levels, decreased IGFBP-4 and IGFBP-5 secretion. IGFBP-5 secretion by BC3H-1 cells was increased by either insulin or IGF-I. Agents which elevate intracellular cAMP concentrations did not increase IGFBP-5 secretion. Additionally, these agents were not synergistic with either insulin or IGF-I. However, indomethacin and progesterone depressed IGFBP-5 secretion by BC3H-1 cells. In summary, there appear to be at least two intracellular signaling mechanisms controlling IGFBP-4 and IGFBP-5 secretion by L6 and BC3H-1 muscle cells. IGFBP secretion by L6 cells is stimulated by both insulin/IGF-I and cAMP-dependent pathways, whereas IGFBP-5 secretion by BC3H-1 cells is stimulated only by the insulin/IGF pathway. IGFBP secretion by both cell lines can be decreased by agents which depress cAMP levels. Our results suggest that two divergent but synergistic pathways modulate IGFBP production and these mechanisms can potentially modulate IGF activity during muscle cell proliferation and differentiation.

The effects of cyclic fatty acid monomers on cultured porcine endothelial cells.

The popularity of polyunsaturated oils used in food applications and preparation continues to appreciate as a result of positive health claims. With polyunsaturated oils inherently more susceptible to oxidative and thermal degradation, the formation of new fatty acid species increases considerably. The presence of one species known as cyclic fatty acid monomers (CFAM) has been detected as a component of many oils subjected to various thermal processes including deep-fat frying. The effect of CFAM on metabolic processes has not been fully characterized. In this study, confluent porcine aortic endothelial cells incorporated CFAM into their polar and nonpolar lipid fractions following a 48-h exposure to 31 and 62 ppm CFAM in the culture medium. Subsequently, the influence of CFAM incorporation on various membrane-dependent physical properties and biochemical processes was investigated. CFAM decreased the lipid packing order of the membrane bilayer core but did not alter the lipid packing order of lipid chain segments at or near the lipid-water interface of the membrane. CFAM led to significant reductions in Ca2+ ATPase activity and monolayer integrity while eliciting a significant increase of prostacyclin synthesis and secretion.

Use of lanthanum to accurately quantify insulin-like growth factor binding to proteins on cell surfaces.

Insulin-like growth factor binding proteins (IGFBPs) are found both associated with cells and in extracellular fluids. Cell-associated IGFBPs increase [125I]-IGF binding to cell monolayers, whereas extracellular (soluble, released) IGFBPs decrease binding. In the current study, we show that either IGFBP-3 or IGFBP-5 are the major forms of IGFBP released from monolayers of human GM10 fibroblasts, T98G glioblastoma cells and forskolin-treated bovine MDBK cells. IGFBPs represent the most abundant [125I]-IGF-I binding site on GM10 and T98G cell monolayers, but 4-17% of the total cell-associated IGFBPs are released from the cell monolayer at 8 degrees C during their quantification. Most of the IGFBPs (> 70%) are released from MDBK cells. Quantitative estimates of [125I]-IGF binding to the cell monolayers are altered because of the ability of the released IGFBPs to reduce the amount of radiolabeled ligand that is available to bind to the cell surface. Lanthanum (La3+) depresses IGFBP release from all three cell types (> 80% for GM10 and T98G cells and > 65% for MDBK cells). The effect was cation specific, noted with La3+ or Zn2+ but not with either Mn2+, Sr2+ or Se3+. The effect was also IGFBP specific; La3+ markedly depressed the release of IGFBP-3 and IGFBP-5, but had less of an effect on IGFBP-2 and IGFBP-4. Concomitant with a decrease in IGFBP-3 and IGFBP-5 release, La3+ caused an increase in [125I]-IGF-I binding to cell-associated IGFBPs and type I IGF receptors. The released soluble IGFBPs have a three- to 20-fold greater affinity (Ka) for [125I]-IGF-I compared to cell-associated IGFBPs. La3+ did not alter the affinity constants of cell-associated IGFBPs. In summary, we have identified a means to prevent loss of IGFBPs from cell monolayers during binding assays. This procedure will be useful in accurately quantifying the levels of IGFBPs on cell monolayers and in determining the role of cell-associated IGFBPs in controlling IGF activity. Retention of cell-associated low affinity IGFBPs may be important in controlling the size of the pericellular IGF pool and in regulating IGF-I access to the type IIGF receptor.

Regulation of myeloid growth and differentiation by the insulin-like growth factor I receptor.

Flow cytometry was used to examine the expression of type I insulin-like growth factor receptors (IGF-IR) on three types of human hematopoietic cells that represent different stages of myeloid lineage development. Both HL-60 (promyeloid) and U-937 (monocytic) cells express abundant IGF-IR protein (> 79% cells positive for the IGF-IR), whereas KG-1 myeloblasts express negligible levels of IGF-IR (< 1% IGF-IR-positive cells). Exogenous IGF-I, IGF-II, and an IGF-I analog that binds poorly to IGF-binding protein-3 (des-IGF-I) increased DNA synthesis of HL-60 and U-937 cells in a dose-dependent (1-25 ng/ml) fashion by 2- to 4-fold in serum-free medium, whereas KG-1 cells did not respond to any of these growth factors. The IGF-induced increase in proliferation of HL-60 promyeloid cells was inhibited by soluble IGF-binding protein-3 (500 ng/ml) when these cells were stimulated with 10 ng/ml of either IGF-I (53 +/- 8%) or IGF-II (59 +/- 8%), but not with des-IGF-I (3 +/- 1%). In contrast, the anti-IGF-IR monoclonal antibody (mAb; alpha IR-3) inhibited the DNA synthesis caused by 10 ng/ml exogenous IGF-I (67 +/- 6%), IGF-II (72 +/- 8%), and des-IGF-1 (82 +/- 9%). Proliferation of KG-1 myeloblasts, however, was neither stimulated by the IGFs nor inhibited by the anti-IGF-IR mAb. In the absence of exogenous IGF-I, the mAb directed against the IGF-IR significantly suppressed basal DNA synthesis of HL-60 promyeloid (72 +/- 5%) and U-937 monocytic (39 +/- 7%) cells, but did not affect DNA synthesis of KG-1 myeloblasts (8 +/- 1%) compared to an isotype-matched control mAb. Similarly, the alpha IR-3 mAb abrogated vitamin D3-induced differentiation of the HL-60 cells into macrophages in serum-free medium, as assessed by expression of the leucam surface protein, CD11b. As the alpha IR-3 mAb inhibits DNA synthesis in the presence and absence of exogenous IGF-I on receptor-bearing cells, but not IGF-IR-negative cells, these data demonstrate that both endocrine and autocrine IGF-I are potent growth factors in human myeloid cells where expression of the surface receptor, rather than the ligand, is the critical control element. More importantly, these data support the hypothesis that autocrine IGF-I may play a significant role in the differentiation of promyeloid cells into macrophages.

Effects of prenatal androgenization and postnatal steroid treatment on growth hormone, insulin-like growth factor I and II, insulin, thyroxine, and triidothyronine concentrations in beef heifers.

A 2 x 3 factorial arrangement of treatments was used to elucidate the mechanism(s) by which prenatal androgenization improves postnatal rate and efficiency of growth and composition of gain in beef heifers. Fifteen control (C) and 15 prenatally androgenized (PA) Angus x Simmental heifers (prenatal treatment, Pretrt) received no (N), estrogen (E), or estrogen and testosterone (ET) implants postnatally (postnatal treatment, Posttrt) to evaluate whether the postpubertal growth response after prenatal androgenization could be induced in prepubertal heifers. Blood was collected from the heifers at 6 +/- 1, 9 +/- 1, and 12 +/- 1 mo of age and analyzed from serum concentrations of growth hormone (GH), IGF-I, IGF-II, insulin, thyroxine (T4), and triiodothyronine (T3). Season of the year had a greater effect on hormone concentrations than either Pretrt or Posttrt, and there were no Pretrt x Posttrt interactions. Prenatal treatment, PA, had no effect on GH; however, Posttrt E and ET increased (P < .001) GH concentrations. Prenatal treatment, PA, increased (P < .05) IGF-I concentrations, and there was a nonsignificant increase (P = .11) in IGF-I concentrations with Posttrt E and ET. Concentrations of IGF-II were unaffected by Pretrt PA; however, they were lower (P < .01) in the Posttrt E and ET groups. Insulin, T4, T3, BW, and ADG were not affected by Pretrt and Posttrt. Concentrations of GH and IGF-I were increased in heifers that received Pretrt PA and(or) Posttrt E and ET in a manner to support improved growth performance; however, BW and ADG were similar. In prepubertal beef heifers, factors in addition to increased GH and IGF-I seem to be necessary for improved growth performance.

Expression of insulin-like growth factor-II and insulin-like growth factor binding proteins during Caco-2 cell proliferation and differentiation.

The components of the insulin-like growth factor (IGF) axis and their roles in regulating proliferation and differentiation of the human colon adenocarcinoma cell line, Caco-2, have been investigated. Caco-2 cells proliferated in serum-free medium at 75% the rate observed in medium containing 10% fetal bovine serum. IGF-I (10 nM) increased Caco-2 cell growth in serum-free medium, but not to the rate seen with serum. Multiple IGF-II mRNA species were produced by Caco-2 cells, but IGF-I mRNA was undetectable. Secretion of radioimmunoassayable IGF-II corresponded with steady-state levels of IGF-II mRNA, neither of which was observed to change markedly over the course of 16 days of Caco-2 cell differentiation. Levels of sucrase-isomaltase mRNA, a marker for enterocytic differentiation, increased 12-fold between days 5 and 16 of culture. Northern blotting of total RNA and ligand blot and immunoblot analyses of serum-free conditioned medium revealed that Caco-2 cells produce several IGF binding proteins (IGFBPs), including IGFBP-2, -3, and -4, as well as a 31,000 M(r) species that was not identified. The pattern of IGFBP secretion changed dramatically during Caco-2 cell differentiation: IGFBP-3 and IGFBP-2 increased 8.5-fold and 5-fold, respectively, whereas IGFBP-4 and the 31,000 M(r) species decreased 43% and 90%. Caco-2 cell clones stably transfected with a human IGFBP-4 cDNA construct exhibited a 60% increase in steady-state level of IGFBP-4 mRNA, and secreted twice as much IGFBP-4 protein as controls. Moreover, IGFBP-4-overexpressing cells proliferated at only 25% the rate of control cells in serum-free medium, in conjunction with a 70% increase in expression of sucrase-isomaltase. In summary, these studies indicate that a complex IGF axis is involved in autocrine regulation of Caco-2 cell proliferation and differentiation.

Macrophages synthesize and secrete a 25-kilodalton protein that binds insulin-like growth factor-I.

Primary (thymus) and secondary (spleen) murine lymphoid tissues express a 25-kDa protein that binds IGF-I. To determine the cellular source of this insulin-like growth factor binding protein (IGFBP), 11 murine or human cell lines representing T, B, and myeloid cells at various stages of differentiation were characterized by IGF-I affinity cross-linkage and Western ligand blotting. Mature myeloid cells, but not T or B cells, secrete a 25-kDa protein that is capable of binding IGF-I. CSF-1-derived bone marrow macrophages also synthesize this 25-kDa IGFBP. Thymic macrophages, which were estimated to secrete 2 ng of binding protein/10(6) cells-h, were used in conjunction with [125I] IGF-I affinity cross-linking to develop a protein binding immunomobility-shift assay to identify which IGFBP is produced by these cells. An anti-IGFBP-4Ab, but not an anti-IGFBP-2 Ab or normal rabbit serum, shifted the [125I] IGF-IGFBP complex to a higher m.w. position, indicating that the single 25-kDa IGFBP is IGFBP-4. Northern blotting confirmed that transcripts for IGFBP-4 as well as IGF-I are expressed in thymic macrophages. A putative 278-bp IGFBP-4 cDNA fragment (residues 341-618) of rat) that contains two unique cysteine residues found only in IGFBP-4 was cloned and sequenced from thymic macrophages. These clones differed from the rat sequence at only six residues (97% homology), and the deduced amino acid sequence from the murine cDNA was identical with that of the rat sequence. Subsequent studies revealed that IGF-I stimulates DNA synthesis in thymic macrophages. However, two different IGF-I analogues differing in the amino-terminus that bind equally well to the IGF-I receptor but poorly to IGFBPs are as effective as IGF-I at 100-fold lower concentrations. These data demonstrate that murine macrophages are a source of a single 25-kDa secreted protein that binds IGF-, that the molecular identity of this protein is IGFBP-4, and that this binding protein may antagonize the extracellular effects of IGF-I.

Effects of cytokines on insulin-like growth factor-binding protein secretion by muscle cells in vitro.

The insulin-like growth factors (IGF-I and IGF-II) stimulate muscle cell proliferation and/or differentiation (depending upon the culture conditions). They also increase IGF-binding protein (IGFBP) levels in muscle cell conditioned media and in some instances there is a direct correlation between the apparent rate of IGFBP secretion and muscle cell proliferation. We have investigated the effect of other cytokines on muscle cell IGFBP conditioned media levels using rat skeletal (L6), mouse myocytes (BC3H-I) and porcine vascular smooth (pVSM) muscle cells in vitro to determine if this relationship is maintained. IGFBP levels in conditioned media (CM) were measured by an [125I]IGF-I binding capacity assay and by western blot analysis. Immunoblots indicated that BC3H-1 and L6 cells secrete IGFBP-5 (31-32,000 M(r)), L6 cells secrete IGFBP-4, and pVSM cells secrete IGFBP-2 (34,000 M(r)). Both L6 and BC3H-1 cells responded to transforming growth factor beta 1 (TGF-beta 1), in a dose-dependent manner, with suppressed conditioned media levels of IGFBP-4 and IGFBP-5 but TGF-beta 1 did not affect IGFBP-2 levels in pVSM cell media. TGF-beta 1 (5 ng/ml) suppressed IGFBP levels (CM [125I]IGF-I binding capacity) in L6 and BC3H-1 cell media by 48% and 61%, respectively. IGFBP-5 levels, in BC3H-1 cell media, were decreased by treatment with either basic fibroblast growth factor (bFGF) or epidermal growth factor (EGF). Neither treatment affected IGFBP-2 levels. In contrast in L6 myoblasts, bFGF increased media levels of IGFBP-4 and IGFBP-5; L6 cells were not responsive to EGF. Insulin increased IGFBP-4 and IGFBP-5 levels in L6 and BC3H-1 cell media. This stimulatory effect was markedly suppressed by either TGF-beta 1 (L6 and BC3H-1 cells) or bFGF (BC3H-1 cells). L6 and BC3H-1 cell CM IGFBP levels were also suppressed 34% and 84% by 5 U/ml thrombin, respectively. The inhibitory activity of thrombin was specific, i.e. reversible by hirudin and was not due to direct IGFBP proteolysis. Since suramin and staurosporine increased media levels of the IGFBP, this suggests that constitutive secretion of TGF-beta 1, bFGF, or EGF might provide a tonic suppressive mechanism for controlling IGFBP secretion. Thus, our results support the conclusion that the secretion of IGFBP-4 and IGFBP-5, but not IGFBP-2, by muscle cells was suppressed by several cytokines. Depressed IGFBP secretion may play a key role in determining muscle cell responsiveness to either the mitogenic or differentiation stimulating activity of the IGFs.

Dexamethasone inhibits mucosal adaptation after small bowel resection.

The present study examined the effects of dexamethasone on mucosal adaptation after massive small bowel resection. Rats underwent 80% jejunoileal resection or a sham operation and received either vehicle or 128 micrograms.kg-1.day-1 sc dexamethasone for 7 days. Dexamethasone infusion resulted in decreased weight, DNA content, and protein content in the duodenojejunal and ileal mucosa in both sham and resected rats. Sucrase, lactase, and maltase activities (all in mumol.g protein-1.min-1) in the duodenojejunal mucosa were elevated by dexamethasone infusion. By contrast, enzyme activities were elevated only in the ileal mucosa of dexamethasone-infused sham-operated rats compared with sham-operated control rats, and dexamethasone did not elevate enzyme activities in resected rats. We further examined whether the inhibitory effects of dexamethasone on mucosal adaptation may be related to changes in either insulin-like growth factor (IGF) or IGF binding protein (BP) serum levels. Serum IGF-I and IGF-II levels were markedly decreased in dexamethasone-infused resected and sham-operated rats. IGF BP-1 serum levels were elevated by dexamethasone treatment with a concomitant depression in serum IGF BP-2 levels. IGF BP-3 levels were lowered by dexamethasone treatment in sham-operated rats and by gut resection, and serum IGF BP-4 levels did not change. These results suggest that the growth-inhibiting effects of dexamethasone in small intestinal mucosa may be partially mediated by decreased serum IGF levels or by alterations in IGF activity associated with changes in serum levels of IGF BPs.

The effects of thyroxine on serum and tissue concentrations of insulin-like growth factors (IGF-I and -II) and IGF-binding proteins in the fetal pig.

We have extensively studied the effect of hypophysectomy on the growth and development of tissues in the fetal pig. However, little is known about the effect of hypophysectomy on tissue levels of insulin-like growth factors I and II (IGF-I and -II) and how these growth factors are affected by T4 replacement. Fetal pigs were hypophysectomized (Hypox) at 70 days of gestation, and pellets containing 15 mg T4 were implanted into the lateral musculature of the hind limb at either 70 or 90 days of gestation. Fetuses were removed at either 90 or 105 days of gestation, respectively. Control (non-Hypox), Hypox, and T4 (Hypox-T4) fetal weights were similar at 90 days, but Hypox-T4 weighted less than control and Hypox fetuses at 105 days. Hypophysectomy decreased levels of serum T4, LH, cortisol, and IGF-I (105 days) when compared with controls. Heart and liver (105 days and 90 days) and fat, muscle, and kidney (90 days) IGF-I levels were lower in Hypox fetuses when compared with controls. Hypophysectomy decreased concentrations of IGF-II in only 105-day fetal kidneys. Hypophysectomy decreased serum levels of IGF binding protein 1 (IGFBP-1) (90 days) and IGFBP-2 (105 days) and increased IGFBP-4 (105 days) in comparison with control. T4 treatment of Hypox fetuses increased serum concentrations of T4 and IGF-I over Hypox levels at both 90 and 105 days gestation. Cortisol levels remained decreased in the T4-treated fetuses. Levels of IGF-I in the heart (90 and 105 days) and liver (90 days) of Hypox fetuses were increased by T4 treatment. T4 did not effect tissue IGF-II levels when compared with Hypox. T4 increased serum IGFBP-1, -2, and -4 levels over Hypox values. We suggest that T4 enhances production of IGF-I (as opposed to IGF-II), which in turn mediates some of T4's capability to enhance tissue development in the fetal pig.

Alterations of serum insulin-like growth factor-I (IGF-I) and IGF-binding proteins (IGFBPs) in swine infected with the protozoan parasite Sarcocystis miescheriana.

The effects of a Sarcocystis miescheriana infection on insulin-like growth factor-I (IGF-I) and insulin-like growth factor binding proteins (IGFBPs) were investigated to determine possible mechanisms of growth retardation in growing pigs. Sixteen pigs averaging 14 kg body weight were divided into 4 groups of 4 pigs each and infected either with 0.5, 1.0, or 3.0 x 10(6) sporocysts of S. miescheriana. Four pigs were retained as non-infected controls; however, they became serologically positive during the course of the infection. Effects also were investigated in 2 groups of 3 pregnant sows. One group was infected with 0.5 x 10(6) sporocysts and the other group was retained as uninfected controls. Body weights of infected growing pigs were depressed as compared to controls following the acute phase 15 d after infection (dai). Serum concentrations of IGF-I dropped significantly (p < 0.05) during the acute phase of infection in all infected groups of growing pigs. Conversely, the amounts of unsaturated serum IGFBPs were elevated significantly (p < 0.05) during the acute phase of infection. Specifically, serum concentrations of IGFBP-1, IGFBP-2, and IGFBP-4 were elevated at this time, as determined by ligand blot analysis. There was no association between growth factor alterations and tissue damage as measured by serum creatinine kinase and aspartate aminotransferase levels. The extent of effects in growing pigs was related to the amount of the original parasite inoculum. During the acute phase of infection 2 of 3 pregnant sows aborted. The third sow went to term, but piglets were stillborn or died within 24 hr. Compared to uninfected controls, serum concentrations of IGF-I in infected pregnant sows were depressed during and after the acute phase of the infection. Levels of unsaturated serum IGFBPs in pregnant sows were not affected. These data suggest that decreased IGF-I levels and/or elevated levels of specific forms of IGFBPs may be a mechanism by which growth is affected in feeder pigs infected with S. miescheriana.

Secretion of insulin-like growth factor II (IGF-II) and IGF-binding protein-2 by intestinal epithelial (IEC-6) cells: implications for autocrine growth regulation.

To identify the factors regulating the proliferation of intestinal epithelium, we examined the effects of various growth factors on [3H] thymidine incorporation into the DNA of IEC-6 cells, an intestinal epithelial cell line derived from rat jejunal crypts. Insulin-like growth factor-I (IGF-I), IGF-II, and insulin stimulated the DNA and protein synthesis of IEC-6 cells in serum-free medium supplemented with transferrin, dexamethasone, and BSA (basal medium). Concentration-response experiments demonstrated that IGF-I is approximately 10 times more potent than IGF-II or insulin in producing 2- to 3-fold stimulations of DNA and protein synthesis by IEC-6 cells. In addition, IEC-6 cells proliferated slowly in the basal medium without any added growth factors. Analysis of medium conditioned by IEC-6 cells by gel filtration chromatography, RIA, HPLC, and N-terminal sequencing revealed that IEC-6 cells synthesize and secrete mature, 7,500 mo wt (M(r)) IGF-II as well as high M(r) forms of IGF-II. In addition, ligand blot, immunoblot, and N-terminal sequence analyses showed that IEC-6 cells produce the 34,000 M(r) IGF-binding protein-2 (IGFBP-2). To determine if IGFBP-2 modulates IGF responses in IEC-6 cells, the IGF-I analogs, Des-(1-3)-IGF-I and [Gln3,Ala4,Tyr15,Leu16]IGF-I, both of which have a reduced affinity for IGFBPs, were tested for their effects on IEC-6 cell proliferation. Both analogs exhibited 10-fold greater potency than IGF-I, presumably because endogenously secreted IGFBPs depress IGF-I binding to cell surface receptors. Finally, purified IGFBP-2 attenuated the DNA synthesis of IEC-6 cells in a dose-dependent manner. We conclude that IGFBP-2 secreted by intestinal epithelial cells is capable of limiting the mitogenic activity of both exogenous and endogenous IGFs by blocking the association of the growth factors with cell surface binding sites. These results further suggest that the growth of IEC-6 cells is modulated by autocrine mechanisms involving IGF-II and IGFBP-2.