Heparin modulates the growth and adherence and augments the growth-inhibitory action of TNF-alpha on cultured human keratinocytes

Previous works suggest the involvement of mast cells in the epithelialization of chronic wounds. Since heparin is a major mediator stored in the secretory granules of mast cells, the purpose of this work was to elucidate the function of heparin in epithelialization using in vitro culture models. For this, low- and high-calcium media in monolayer and epithelium cultures of keratinocytes were used. Also, an assay based on keratinocyte adherence onto plastic surface was used as well. Heparin (0.02-200 microg/ml) inhibited keratinocyte growth in a non-cytotoxic and dose-dependent manner in low- and high-calcium media, Keratinocyte-SFM and DMEM, in the absence of growth factors and serum. Also, heparin inhibited the growth of keratinocyte epithelium in the presence of 10% fetal calf serum and DMEM. Instead, in the presence of Keratinocyte-SFM and growth factors, heparin at 2 microg/ml inhibited the growth by 18% but at higher heparin concentrations the inhibition was reversed to baseline. TNF-alpha is another preformed mediator in mast cell granules and it inhibited keratinocyte growth in monolayer and epithelium cultures. Interestingly, heparin at 2-20 microg/ml augmented or even potentiated this growth-inhibitory effect of TNF-alpha. The association of TNF-alpha with heparin was shown by demonstrating that TNF-alpha bound tightly to heparin-Sepharose chromatographic material. However, heparin could not augment TNF-alpha-induced cell cycle arrest at G0/G1 phase or intercellular adhesion molecule-1 expression in keratinocytes. In the cell adherence assay, heparin at 2 microg/ml inhibited significantly by 12-13% or 33% the adherence of keratinocytes onto the plastic surface coated with fibronectin or collagen, respectively, but this inhibition was reversed back to baseline at 20 or 200 microg/ml heparin. Also, heparin affected the cell membrane rather than the protein coat on the plastic surface. In conclusion, heparin not only inhibits or modulates keratinocyte growth and adherence but it also binds and potentiates the growth-inhibitory function of TNF-alpha.     

Electron microscopic analysis of glucose-induced endothelial damage in primary culture: possible mechanism and prevention

We previously reported that high glucose treated cultured endothelial cells (ECs) showed intercellular gaps by transmission electron microscopy (TEM). These gaps were abrogated with insulin and/or heparin treatment. Our aims were to assess the severity of injury in ECs treated with high glucose for variable duration, and to further study the protective effects of insulin and/or heparin. Cells were also treated with L-buthionine sulfoximine (BSO), a glutathione inhibitor, to help understand the mechanism of high glucose injury. Primary porcine ECs were treated with high glucose (30 mM) for 2, 6 or 10 days; and glucose plus insulin (1 U/ml), glucose plus heparin (5 microg/ml), glucose plus insulin plus heparin for 6 days. ECs were treated with BSO (0.001-0.05 mM) for 2 days. Pellets from trypsinized cells were processed for TEM. High glucose treatment revealed apoptosis or necrosis showing variable cell size, abnormal nuclei, condensation of nuclear chromatin, few mitochondria, cell membrane disruption and needle-shaped structures. Changes increased with duration of exposure. In high glucose plus heparin or insulin treated cultures at least one-half of the cells appeared normal. Most ECs were intact when treated with high glucose plus insulin plus heparin. BSO treatment showed dose-dependent changes with low doses showing apoptosis whereas higher doses revealed necrosis similar to high glucose treatment for 6 or 10 days. High glucose-induced EC injury increased with duration of exposure. These data demonstrate that high glucose injury resembles that of BSO treatment, suggesting that glutathione depletion may be involved in EC injury. Insulin and/or heparin protect against high glucose-induced injury.     

Insulin potentiates EGFR activation and signaling in fibroblasts

Insulin is an essential hormone for cell growth and potentiates the mitogenic actions of multiple growth factors, including EGF. While potentiation has been shown to be mediated by the upregulation of the cyclin/CDK system, the upstream mechanisms of such synergy have not been elucidated. Our study has examined whether insulin could mediate synergy by enhancing early signaling events of the EGF receptor (EGFR). Tyrosine phosphorylation at the cell periphery of confluent Swiss 3T3 fibroblasts induced by EGF was potentiated by insulin within 2 min of stimulation. Insulin potentiation of EGF-mediated phosphorylation of the EGFR occurred 2 min after stimulation. EGFR transactivation by insulin was not observed. In addition, downstream mitogenic signaling events including ERK1/2 activation and Elk-1 phosphorylation were enhanced in response to insulin and EGF coadministration. This study shows mitogenic synergy between insulin and EGF can occur at the earliest signaling event, receptor phosphorylation, and independent of transactivation.     

Effect of heparin on the production of matrix metalloproteinases and tissue inhibitors of metalloproteinases by human dermal fibroblasts

The influence of heparin and a heparin fragment devoid of anticoagulant activity on the production of matrix metalloproteinases and tissue inhibitors of metalloproteinases by human dermal fibroblasts was studied. Doses (0.1-400 microg/ml) responses were performed and data obtained were similar whatever heparin or fragment was used. The basal expression of collagenase by fibroblasts decreased quasi-linearly with increasing doses of heparins from 1 to 400 microg/ml. TIMP-1 levels were not affected by supplementing serum free culture medium with heparins. On the contrary, at low concentration, i.e. 1-10 microg/ml, heparins stimulated the secretion of both 72-kDa gelatinase (1.4-1.6-fold) and particularly TIMP-2 (>4-fold). At high doses, MMP-2 and TIMP-2 production by fibroblasts returned to basal levels. These results suggested that the local concentration of heparin released by mast cells could be instrumental in modulating fibroblast growth and proteolytic phenotype.     

Serum-free cell culture on insulin-immobilized porous collagen beads

Insulin or albumin was immobilized on collagen beads using water-soluble carbodiimide. Adhesion of STO mouse fibroblast cells onto the beads decreased with increasing the amount of immobilized proteins. Growth of the cells was remarkably accelerated on the insulinimmobilized collagen beads, which can be used for serum-free cell culture. The growth acceleration became larger with increasing the amount of immobilized insulin, while it became smaller with increasing the amount of immobilized albumin. In addition, the immobilized insulin more strongly accelerated the cell growth than free insulin plus collagen beads. (c) 1995 John Wiley & Sons, Inc.     

Hibernation, reversible cell growth inhibition by epigallocatechin-3-O-gallate

Epigallocatechin-3-O-gallate (EGCg) and related polyphenolic compounds found in tea are known to have antioxidative activities. However, they also have pro-oxidative activities such as generation of hydrogen peroxide. In this report, we investigated the effect on cells and showed the potential usage of EGCg in cell preservation. H(2)O(2) was generated from EGCg at concentrations of more than 300 microg/mL for 6 h at 37 degrees C, and high cytotoxicity for L929 cells were shown. In contrast, in the presence of 1 microg/mL catalase, the amount of generated H(2)O(2) was significantly low and cytotoxicity decreased markedly. This indicates that catalase eliminated H(2)O(2) generated by degradation of EGCg. Although H(2)O(2) generation was prevented, L929 cell proliferation was slightly inhibited in proportion to the concentrations of EGCg. L929 was exposed able to be 300 microg/mL to EGCg and 1 microg/mL catalase for maximum 18 days. EGCg inhibited the growth of L929 cells, and cell proliferation was restarted immediately after medium change for removing EGCg. We concluded that EGCg had a reversible growth inhibition when H(2)O(2) was eliminated from cell cultures.     

Bioactive microarrays immobilized on low-fouling surfaces to study specific endothelial cell adhesion

With the aim to study how to modulate the specific endothelial cell patterning and responses on biomaterials surfaces, bioactive microarrays were developed and validated for specific cell patterning. These microarrays were made of low-fouling surfaces, that prevent nonspecific cell adhesion, bearing bioactive molecules at given known locations by presenting specific ligands to cell receptors. Arrays of bioactive molecules (RGD, REDV, and SVVYGLR sequences and vascular endothelial growth factor (VEGF)) were immobilized on a carboxy-methyl-dextran low-fouling surface and were exposed to human endothelial cells and fibroblasts to screen for the effect of bioactive spot molecular composition on cell adhesion. Endothelial cells only were sensitive to RGD peptide co-immobilized with REDV or SVVYGLR sequences: they induced a reduction in cell spreading and a loss of actin stress fibers. RGD co-immobilized with VEGF also resulted in the reorganization of actin filaments and focal points in endothelial cells. Combination of RGD with these endothelial cell-selective biomolecules did not elicit a strong adhesion phenotype but rather one characteristic of migrating cells.     

Characterization of the growth of murine fibroblasts that express human insulin receptors. I. The effect of insulin in the absence of other growth factors

The effect of insulin on the growth of murine fibroblasts transfected with an expression vector containing human insulin receptor cDNA (NIH 3T3/HIR) and the parental cells (NIH/3T3) was characterized. Insulin in the absence of other mitogens increased the rate of incorporation of thymidine into NIH 3T3/HIR cells with a half-maximal response occurring at an insulin concentration of 35 ng/ml and a maximal response that was equivalent to that elicited by 10% fetal calf serum. The thymidine incorporation rate was increased by 12 h, was maximal at approximately 16 h, and returned to basal rates at 24 h after the addition of insulin. Insulin induced a maximum of 65% of cells to incorporate thymidine. The increased DNA synthesis was accompanied by net growth. Addition of insulin to the NIH 3T3/HIR cells resulted in increased DNA content with a half-maximal response occurring at approximately 30 ng/ml insulin and a maximal response equivalent to that elicited by serum. An increase in cell number detected after the addition of insulin to the NIH 3T3/HIR suggests that the cells had progressed through mitosis. Insulin did not increase the rate of thymidine incorporation, DNA content, or number of the parental NIH 3T3 cells. These data show that insulin, in the absence of a second mitogen, is able to induce NIH 3T3/HIR fibroblasts to traverse the cell cycle.     

Insulin stimulation of glucose transport and metabolism in a human Wilms' tumor-derived myoblast-like cell Line: Modulation of hormone effects by glucose deprivation

The effects of insulin and glucose on parameters of metabolism were investigated in myoblast-like (MBL) cells, a human myoblast-like cell line derived from a Wilms' tumor. Insulin responses were studied after 4 hr pre-incubation in serum free media, with or without 5 mM glucose. Insulin was added during the last 2 hr. Glucose starvation markedly increased basal glucose transport (measured as 2-deoxyglucose uptake) as well as the net uptake of [¹⁴C]glucose and [¹⁴C]glucose incorporation into glycogen. Insulin stimulated net glucose uptake and incorporation into glycogen in a dose-dependent manner in glucose-fed and starved cells. These insulin responses were markedly enhanced in glucose-starved cells. Insulin accelerated 2-deoxyglucose transport in glucose-fed cells but did not further stimulate basal glucose transport in glucose-deprived cells. Insulin increased the incorporation of [³H]leucine into protein in glucose-fed or -starved MBL cells equally. The dose of insulin required for half-maximal insulin responses was similar for all parameters studied. Cycloheximide did not prevent the increased basal glucose incorporation in glucose-starved cells, but markedly inhibited the insulin response, while in glucose-fed cells, cycloheximide stimulated basal glucose incorporation. We conclude that MBL cells resemble fibroblasts in their insulin-independent stimulation of glucose transport in response to glucose-deprivation; when provided with glucose, they respond to insulin like fibroblasts. However, after brief glucose-starvation, the stimulated glucose transport system is no longer insulin-responsive in MBL cells, while pathways leading to the synthesis of macromolecules demonstrate preserved or enhanced stimulation by insulin, suggesting that these cells may serve as models to study the regulation of receptor-response coupling by the metabolic milieu.     

Membrane bound and cellular cationic changes associated with insulin stimulation of cultured cells

Insulin was employed as a stimulant in our continuing investigations of the molecular mechanisms involved in the coordinate control of cellular metabolism and growth. Incubation of chicken embryo fibroblasts for 16 hours in media containing 0-0.1 U insulin/ml resulted in a 17-fold increase in the rate of 3H-thymidine incorporation into DNA. Concomitantly, there were graded increases in intracellular K+ (14%) AND Mg2+ (22%) and no significant change in Ca2+. These changes in cation content occurred within 10 to 30 minutes and preceded the changes in 3H-thymidine incorporation. Insulin produced a consistent graded decrease in externally bound Mg2+ and Ca2+ and a concomitant increase in bound Na+ and K+ with no significant change in the rates of K+ and Mg2+ efflux. The results are consistent with the concept of Mg2+ as a second messenger for insulin action, as well as with the more general hypothesis that Mg2+ is the centtral agent in the coordinate control of metabolism and growth in animal cells.     

Regulation of insulin/insulin-like growth factor-1 signaling by proteasome-mediated degradation of insulin receptor substrate-2

Insulin and insulin-like growth factor-1 (IGF-1) regulate metabolism and body growth through homologous receptor tyrosine kinases that phosphorylate the insulin receptor substrate (IRS) proteins. IRS-2 is an important IRS protein, as it mediates peripheral insulin action and beta-cell survival. In this study, we show that insulin, IGF-1, or osmotic stress promoted ubiquitin/proteasome-mediated degradation of IRS-2 in 3T3-L1 cells, Fao hepatoma, cells and mouse embryo fibroblasts; however, insulin/IGF-1 did not promote degradation of IRS-1 in 3T3-L1 preadipocytes or mouse embryo fibroblasts. MG132 or lactacystin, specific inhibitors of 26S proteasome, blocked insulin/IGF-1-induced degradation of IRS-2 and enhanced the detection of ubiquitinated IRS-2. Insulin/IGF1-induced ubiquitination and degradation of IRS-2 was blocked by inhibitors of phosphatidylinositol 3-kinase (wortmannin or LY294002) or mTOR (rapamycin). Chronic insulin or IGF-1 treatment of IRS-1-deficient mouse embryo fibroblasts inhibited IRS-2-mediated activation of Akt and ERK1/2, which was reversed by lactacystin pretreatment. By contrast, IRS-1 activation of Akt and ERK1/2 was not inhibited by chronic insulin/IGF-1 stimulation in IRS-2-deficient mouse embryo fibroblasts. Thus, we identified a novel negative feedback mechanism by which the ubiquitin/proteasome-mediated degradation of IRS-2 limits the magnitude and duration of the response to insulin or IGF-1.     

Differentiated fibroblastic progenies of human embryonic stem cells for toxicology screening

Immortalized cell lines and live animal models are commonly used for cytotoxicity screening of biomedical devices and materials. However, these assays poorly reflect human physiology and have numerous other disadvantages. An alternative may be to utilize differentiated fibroblastic progenies of human embryonic stem cells (hESC) for in vitro toxicology screening. These were generated through random spontaneous differentiation within standard culture media, over several passages. The cytotoxic response of the differentiated hESC fibroblastic progenies (pH9) to mitomycin C was observed to be not only very similar to the L929 cell line, but was, in fact, more sensitive. At an initial seeding density of 1000 cells/well (0.33 cm(2)), the proliferation index was observed to decrease 19.0% from 1.638 to 1.326 for the L929 cell line, as the dosage of mitomycin C was gradually increased from 0 to 1.54 microg/mL. By contrast, pH9 displayed a corresponding 40.5% drop in proliferation index from 3.713 to 2.209. At a higher seeding density of 2000 cells/well (0.33 cm(2)), the proliferation index was observed to decrease 27.0% from 1.213 to 0.885 for the L929 cell line, whereas pH9 displayed a corresponding 43.7% drop in proliferation index from 3.711 to 2.091. Hence, it is apparent that pH9 exhibited a more sensitive dose-response to mitomycin C compared to L929, which could be advantageous for cytotoxicity screening assays. Additionally, this study also demonstrated that a highly purified and well-defined phenotypic population of differentiated hESC progenies is not necessary for high reproducibility and accuracy in cytotoxic response.     

Effects of sperm treatments on the in vitro development of bovine oocytes in semidefined and defined media

Bovine in vitro matured oocytes were inseminated with frozen-thawed spermatozoa prepared by A) swim-up through Fert-TALP supplemented with hyaluronic acid (HYA, 1 mg/ml), heparin (5.0 microg/ml) and bovine serum albumin (BSA, 6 mg/ml) or B) washing by centrifugation in modified Brackett-Oliphant medium (mBO) supplemented with 10 mM caffeine-sodium benzoate. For Method A, in vitro fertilization (IVF) was performed in Fert-TALP supplemented with 6 mg/ml BSA, 5.0 microg/ml heparin, 20 microM D-penicillamine, 10 microM hypotaurine and 1 microM epinephrine. For Method B it was performed in mBO medium supplemented with 10 mg globulin-free BSA/ml and 10 microg heparin/ml. Presumptive zygotes were cultured in 1 of 3 culture media: 1) BSAITS - TCM 199 supplemented with 10 mg/ml BSA and ITS (5 microg/ml insulin, 5 microg/ml transferrin, and 5 ng/ml sodium selenite); 2) BECM - bovine embryo culture medium; and 3) BECM supplemented with ITS. Altogether, a significantly higher proportion of oocytes developed to the blastocyst stage after insemination with spermatozoa prepared by Method A than by Method B (17.9 vs 7.1%, respectively; P < 0.001). For Method A, the cleavage rate and the proportion of zygotes with >2 cells 48 h after insemination did not differ significantly between any of the 3 culture media assayed, but blastocyst formation was significantly stimulated in BSAITS and BECMITS compared with that in BECM (20.7 and 22.1% vs 10.7%, respectively; P < 0.05). For Method B, the cleavage rate and the proportion of zygotes with >2 cells were significantly lower in BSAITS than in BECM and BECMITS (56.4 and 28.7% vs 71.6 and 42.1%; and 70.2 and 51.1%, respectively; P < 0.05). However, no significant differences were recorded in blastocyst development rates between any of the culture media assayed (6.4 to 7.4%; P > 0.05).     

Nuclear transport in 3T3 fibroblasts: effects of growth factors, transformation, and cell shape

Nucleocytoplasmic transport of fluorescent-labeled macromolecules was investigated in transformed and nontransformed 3T3 fibroblasts. Insulin and epidermal growth factor enhanced transport three-fold after 1-2-h incubation with nontransformed adhering fibroblasts; no enhancement of transport was observed for spherical unattached fibroblasts. The concentration of growth factor for maximal enhancement was 3-10 nM. Nuclear transport for Kirsten murine sarcoma virus-transformed BALB/c 3T3 fibroblasts, however, was maximally enhanced before addition of growth factors; addition of insulin or epidermal growth factor causes no additional transport enhancement. Transformation also minimizes cell shape effects on macromolecular nuclear transport. These results provide evidence that protein growth factors and oncogenic transformation may use a similar mechanism for activation of nuclear transport.     

The fusion glycoprotein of human respiratory syncytial virus facilitates virus attachment and infectivity via an interaction with cellular heparan sulfate

Human respiratory syncytial virus (RSV) F glycoprotein (RSV-F) can independently interact with immobilized heparin and facilitate both attachment to and infection of cells via an interaction with cellular heparan sulfate. RSV-glycosaminoglycan (GAG) interactions were evaluated using heparin-agarose affinity chromatography. RSV-F from A2- and B1/cp-52 (cp-52)-infected cell lysates, RSV-F derived from a recombinant vaccinia virus, and affinity-purified F protein all bound to and were specifically eluted from heparin columns. In infectivity inhibition studies, soluble GAGs decreased the infectivity of RSV A2 and cp-52, with bovine lung heparin exhibiting the highest specific activity against both A2 (50% effective dose [ED(50)] = 0.28 +/- 0.11 microg/ml) and cp-52 (ED(50) = 0.55 +/- 0. 14 microg/ml). Furthermore, enzymatic digestion of cell surface GAGs by heparin lyase I and heparin lyase III but not chondroitinase ABC resulted in a significant reduction in cp-52 infectivity. Moreover, bovine lung heparin inhibited radiolabeled A2 and cp-52 virus binding up to 90%. Taken together, these data suggest that RSV-F independently interacts with heparin/heparan sulfate and this type of interaction facilitates virus attachment and infectivity.     

Cell growth on immobilized cell-growth factor; 4: Interaction of fibroblast cells with insulin immobilized on poly(methyl methacrylate) membrane.

Insulin was immobilized on the surface-hydrolyzed poly(methyl methacrylate) membrane and the growth acceleration of mouse fibroblast cells, STO, by the immobilized insulin was investigated. It was found that insulin remains immobilized on the surface of nonbiodegradable membrane and interacts specifically with receptors existing on the biological membrane of fibroblast cells. The growth acceleration by immobilized insulin was enhanced by introduction of a spacer arm between insulin and the immobilization matrix. The amount of receptor proteins present on the biological membrane of fibroblast cells after culturing with insulin, immobilized on nonbiodegradable polymer membrane, was much higher than that after culturing with free insulin, implying the suppression of down-regulation in the case of immobilized insulin.     

Mitogenic stimulation of human breast cancer cells in a growth factor-defined medium: synergistic action of insulin and estrogen

The cooperative action of 17 beta-estradiol (E2) and polypeptide growth factors in stimulating proliferation of human breast cancer cells in vitro was investigated. To prevent background estrogenic stimulation, only phenol red-free media were used. When cultured in media supplemented with steroid-stripped serum in which all polypeptide growth factor activity had been chemically inactivated, MCF7 cells were unable to proliferate and became virtually quiescent. In the additional presence of insulin, epidermal growth factor (EGF), and E2, however, cells proliferated as rapidly as did cells cultured in media supplemented with fetal calf serum. Analysis by DNA flow cytometry showed that in the absence of external growth factors, MCF7 cells became arrested predominantly in the G1/G0 phase of the cell cycle. Upon addition of insulin in combination with EGF and E2, however, cells reentered the cell cycle with a high degree of synchrony. When added alone, E2 induced only slight mitogenic effects under these growth factor-defined conditions. In contrast, this steroid induced optimal proliferation in conventional steroid-stripped serum, which in itself contained considerable mitogenic activity. Insulin (at 10 micrograms/ml) was the most potent stimulator of MCF7 cell proliferation under growth factor-defined conditions, resulting in a more than sixfold increase in cell number after 96 hours. Other growth factors such as platelet-derived growth factor (PDGF), transforming growth factor beta (TGF beta), and EGF had little effect by themselves and only slightly influenced insulin-induced proliferation. At suboptimal concentrations of insulin (10-100 ng/ml), however, strong synergism was observed between E2 and insulin in inducing MCF7 proliferation. Using the CG5 cell line, a highly E2-sensitive MCF7 variant, synergism with E2 was already observed at 1 ng/ml insulin. It is concluded that MCF7 cells require insulin (or insulin-like growth factors) for proliferation. At suboptimal insulin concentrations, E2 acts synergistically with insulin, possibly by inducing autocrine production of polypeptide growth factors by these cells.     

Insulin-like growth factor I and insulin rapidly increase casein kinase II activity in BALB/c 3T3 fibroblasts

We have tested whether growth factors added to serum-deprived BALB/c 3T3 fibroblasts alter the casein kinase II activity measured in cell extracts. A rapid phosphocellulose chromatography method was developed that provides a 40-fold partial purification of casein kinase II activity assayed with the specific substrate peptide Arg-Arg-Glu-Glu-Glu-Thr-Glu-Glu-Glu. Using this technique, kinase activity is stimulated 1.6-2.5-fold when isolated from fibroblasts treated with insulin or insulin-like growth factor I (IGF-I). The activated kinase activity exhibits the specific properties of casein kinase II such as the ability to utilize [gamma-32P]GTP as phosphate donor and marked inhibition by low concentrations of heparin. Activation of casein kinase II appears specific for these hormones because epidermal growth factor and platelet-derived growth factor have no effect on the kinase activity when added to fibroblasts under conditions where they markedly stimulate [3H]thymidine incorporation into DNA. Increases of casein kinase II activity by insulin and IGF-I were detected within 1 min of their addition to cell cultures. IGF-I is more potent in stimulating casein kinase II than insulin in mouse fibroblasts. These results demonstrate that casein kinase II is a selective target for insulin and IGF-I action in BALB/c fibroblasts, consistent with the hypothesis that this kinase plays a role in cellular signaling by these hormones.     

Coupling of insulin-responsive glucose transport to receptors for insulin-like growth factor 1 in primary human fibroblasts

We have recently described an insulin-resistant patient with leprechaunism (leprechaun G.) having a homozygous leucine----proline mutation at amino acid position 233 in the alpha-chain of the insulin receptor. The mutation results in a loss of insulin binding to cultured fibroblasts. Fibroblasts from the patient and control individuals were used to quantify the stimulation of 2-deoxyglucose uptake by insulin and insulin-like growth factor 1 (IGF-1). Insulin hardly stimulates basal 2-deoxyglucose uptake in the patient's fibroblasts whereas in control fibroblasts the uptake of 2-deoxyglucose is stimulated by insulin approximately 1.7 times. In contrast, IGF-1 stimulates hexose uptake in the patient's fibroblasts 1.8 times, a similar value to that obtained by stimulation of control fibroblasts with insulin or IGF-1. With both types of fibroblasts, maximal IGF-1 response is reached at about 10 nM IGF-1, the ED50 being approximately 4 nM. The results indicate that the insulin responsive glucose transport in primary fibroblasts is functionally linked to the receptor for IGF-1. Insulin binds with an approximately 200-fold lower affinity to IGF-1 receptors, compared to homologous IGF-1 binding. As an insulin concentration of 10 microM is unable to give maximal stimulation of glucose uptake in the patient's fibroblasts, which is already seen with 10 nM IGF-1, it seems that occupation of IGF-1 receptors by insulin on the patient's cells is less efficient at stimulating hexose uptake compared to homologous activation.