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PubMed2000

Determination of non-transferrin-bound iron in genetic hemochromatosis using a new HPLC-based method.

BACKGROUND/AIMS: Non-transferrin-bound iron may play a major pathogenic role in iron overload diseases due to its high hepatic uptake and potential damaging effect. The aim of this study was to evaluate the relevance of measuring serum non-transferrin-bound iron levels in genetic hemochromatosis using a new high performance liquid chromatography-based method. METHODS: This method includes a presaturation step of transferrin with cobalt(II) in order to avoid secondary deplacement of non-transferrin-bound iron toward transferrin during the assay. Six genetic hemochromatotic patients were followed serially during venesection treatment. RESULTS/CONCLUSIONS: The results indicate: (i) that this new method permits detection of non-transferrin-bound iron when transferrin is not fully saturated, (ii) that non-transferrin-bound iron levels persist almost until the completion of treatment, (iii) that non-transferrin-bound iron levels are well correlated with transferrin saturation for a given patient, and (iv) that despite some individual variations, a transferrin saturation value lower than 35% usually corresponds to the disappearance of non-transferrin-bound iron.

Adult↗
PubMed2003

Mechanisms and regulation of transferrin and iron transport in a model blood-brain barrier system.

For peripheral iron to reach the brain, it must transverse the blood-brain barrier. In order for the brain to obtain iron, transferrin receptors are present in the vascular endothelial cell to facilitate movement of transferrin bound iron into the brain parenchyma. However, a number of significant voids exist in our knowledge about transport of iron into the brain. These gaps in our knowledge are significant not only because iron is an essential neurotrophic factor but also because the system for delivery of iron into the brain is being viewed as an opportunity to circumvent the blood-brain barrier for delivery of neurotoxins to tumors or trophic factors in neurodegenerative diseases. In this study, we have used fluorescein-transferrin-59Fe in a bovine retinal endothelial cell culture system to determine the mechanism of transferrin-iron transport and to test the hypothesis that the iron status of the endothelial cells would influence iron transport. Our results indicated that iron is transported across endothelial cells both bound to and not bound to transferrin. The ratio of non-transferrin-bound iron to transferrin-bound iron transported is dependent upon the iron status of the cells. Blocking acidification of endosomes led to a significant decrease in transport of non-transferrin-bound iron but not transferrin-bound iron. Blocking pinocytosis had no effect on either transferrin or iron transcytosis. These results indicate that there is both transferrin-mediated and non-transferrin-mediated transcytosis of iron and that the process is influenced by the iron status of the cells. These data have considerable implications for common neurodegenerative diseases that are associated with excess brain iron accumulation and the numerous neurological complications associated with brain iron deficiency.

Animals↗
PubMed2003

Transferrin receptor 2: a new molecule in iron metabolism.

Transferrin receptor 1 (TfR1) which mediates uptake of transferrin-bound iron, is essential for life in mammals. Recently, a close homologue of human transferrin receptor 1 was cloned and called transferrin receptor 2 (TfR2). A similar molecule has been identified in the mouse. Human transferrin receptor 2 is 45% identical with transferrin receptor 1 in the extracellular domain, but contains no iron responsive element in its mRNA and is apparently not regulated by intracellular iron concentration nor by interaction with HFE. Transferrin receptor 2, like transferrin receptor 1, binds transferrin in a pH-dependent manner (but with 25 times lower affinity) and delivers iron to cells. However, transferrin receptor 2 distribution differs from transferrin receptor 1, increasing in differentiating hepatocytes and decreasing in differentiating erythroblasts. Expression of both receptors is cell cycle dependent. Mutations in the human transferrin receptor 2 gene cause iron overload disease, suggesting it has a role in iron homeostasis.

Animals↗
PubMed2001

Acquisition of iron from human transferrin by Porphyromonas gingivalis: a role for Arg- and Lys-gingipain activities.

Porphyromonas gingivalis, a key causative agent of adult periodontitis, is known to produce a variety of virulence factors including proteases. The aim of this study was to evaluate the participation of Arg- and Lys-gingipain activities of P. gingivalis in the acquisition of iron from human transferrin and its subsequent utilization in growth. Iron-saturated transferrin was found to support the long-term growth of P. gingivalis. Our results indicated that P. gingivalis does not produce siderophore and does not possess ferric reductase and transferrin-binding activities. Incubating transferrin with P. gingivalis resulted in degradation of the protein, a step that may be critical for the acquisition of iron from transferrin. Spontaneous and site-directed mutants of P. gingivalis deficient in one or several proteases were used to demonstrate the key role of specific enzymes in degradation of transferrin and subsequent utilization for growth. The lack of both Arg- and Lys-gingipain activities (mutants M1 and KDP128) was associated with an absence of degradation of transferrin and the incapacity of bacteria to grow in the presence of transferrin as the sole source of iron. It was also found that the Lys-gingipain activity is more critical than the Arg-gingipain activity since the mutant KDP112 (deficient in Arg-gingipain A and B) could grow whereas the mutant KDP129 (deficient in Lys-gingipain) could not. The fact that growth of mutant KDP112 was associated with a lower final optical density and a generation time much longer compared with the parent strain suggests that the Arg-gingipain activity also participates in the acquisition of iron from transferrin. Selected inhibitors of cysteine proteases (TLCK, leupeptin and cathepsin B inhibitor II) were tested for their capacity to reduce or inhibit the growth of P. gingivalis under different iron conditions. All three inhibitors were found to completely inhibit growth of strain ATCC 33277 in a medium supplemented with transferrin as the source of iron. The inhibitors had no effects when the bacteria were grown in a medium containing hemin instead of transferrin. The ability of P. gingivalis to cleave transferrin may be an important mechanism for the acquisition of iron from this protein during periodontitis.

Adhesins, Bacterial↗
PubMed1998

Ferric citrate uptake by cultured rat hepatocytes is inhibited in the presence of transferrin.

Diseases associated with iron overload occur worldwide. In subjects suffering from these conditions, transferrin is likely to be fully saturated and excess plasma iron must be complexed to other molecules. Consequently, the liver, which is the major site of iron storage, will be presented with iron in both transferrin-bound and non-transferrin-bound forms and these forms may compete for uptake by hepatocytes. The endogenous low-molecular-mass iron chelator, citrate, is considered to be a major contributing molecule to non-transferrin iron transport. This study was conducted to investigate the effects of transferrin on the uptake of citrate and iron citrate by hepatocytes in culture. Rat hepatocytes were incubated with 100 microM [14C]citrate and 1.0 microM 55Fe in the presence or absence of various forms of transferrin. Binding and internalisation of both citrate and iron were inhibited in a dose-dependent manner with increasing concentration of diferric transferrin, with iron uptake decreasing to less than 5% of control values. Apotransferrin was markedly more effective in blocking citrate and iron uptake, reaching the same levels of inhibition at a 15-fold lower concentration of protein. The binding of citrate to the cell membrane was not affected significantly by changing the iron saturation of transferrin but internalisation decreased with decreasing saturation. In contrast, both the binding and internalisation of iron decreased with decreasing saturation. Incubations carried out using 55Fe-labelled citrate in the presence of 59Fe-labelled diferric transferrin indicated that citrate-mediated iron binding by the cells decreased with increasing diferric transferrin concentrations but the citrate iron was not replaced by iron from transferrin during the 15-min incubation period. Instead, total iron uptake decreased. These data suggest that citrate-mediated iron uptake by hepatocytes shares at least one common pathway with transferrin-mediated iron uptake.

Animals↗
PubMed2006

Radioprotective effect of transferrin targeted citicoline liposomes.

The high level of expression of transferrin receptors (Tf-R) on the surface of endothelial cells of the blood-brain-barrier (BBB) had been widely utilized to deliver drugs to the brain. The primary aim of this study was to use transferrin receptor mediated endocytosis as a pathway for the rational development of holo-transferrin coupled liposomes for drug targeting to the brain. Citicoline is a neuroprotective agent used clinically to treat for instance Parkinson disease, stroke, Alzheimer's disease and brain ischemia. Citicoline does not readily cross the BBB because of its strong polar nature. Hence, citicoline was used as a model drug. (Citicoline liposomes have been prepared using dipalmitoylphosphatidylcholine (DPPC) or distearoylphosphatidylcholine (DSPC) by dry lipid film hydration-extrusion method). The effect of the use of liposomes composed of DPPC or DSPC on their citicoline encapsulation efficiency and their stability in vitro were studied. Transferrin was coupled to liposomes by a technique which involves the prevention of scavenging diferric iron atoms of transferrin. The coupling efficiency of transferrin to the liposomes was studied. In vitro evaluation of transferrin-coupled liposomes was performed for their radioprotective effect in radiation treated cell cultures. In this study, OVCAR-3 cells were used as a model cell type over-expressing the Tf-R and human umbilical vein endothelial cells (HUVEC) as BBB endothelial cell model. The average diameter of DPPC and DSPC liposomes were 138 +/- 6.3 and 79.0 +/- 3.2 nm, respectively. The citicoline encapsulation capacity of DPPC and DSPC liposomes was 81.8 +/- 12.8 and 54.9 +/- 0.04 microg/micromol of phospholipid, respectively. Liposomes prepared from DSPC showed relatively better stability than DPPC liposomes at 37 degrees C and in the presence of serum. Hence, DSPC liposomes were used for transferrin coupling and an average of 46-55 molecules of transferrin were present per liposome. Free citicoline has shown radioprotective effect at higher doses tested. Interestingly, encapsulation of citicoline in pegylated liposomes significantly improved the radioprotective effect by 4-fold compared to free citicoline in OVCAR-3 but not in HUVEC. Further, citicoline encapsulation in transferrin-coupled liposomes has significantly improved the radioprotective effect by approximately 8-fold in OVCAR-3 and 2-fold in HUVEC cells with respect to the free drug. This is likely due to the entry of citicoline into cells via transferrin receptor mediated endocytosis. In conclusion, our results suggest that low concentrations of citicoline encapsulated in transferrin-coupled liposomes could offer therapeutic benefit in treating stroke compared to free citicoline.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed1995

The heterogeneous distribution of brain transferrin.

Immunocytochemistry with antisera to transferrin has often been used to identify oligodendroglia in tissue sections and cultures, but reaction product also occurs in blood vessel walls and nerve cells. There is considerable species variation. Serum transferrin is largely biosynthesized in the liver, and its established physiological role is the transport of iron to tissue sites and delivery of the metal to the interior of cells that have transferrin receptors on their surfaces. In sections of the central nervous system, the visualization of iron and transferrin generally does not coincide, and transferrin may have importance to normal brain function beyond iron transport. For a comparative analysis of transferrin in rabbit and rat brain, polyclonal antisera were raised against purified serum transferrins of these species. The antisera were used for transferrin immunocytochemistry on vibratome sections and for immunochemical detection on electroblots. Transferrin immunocytochemistry and iron histochemistry were compared. The electrophoretic separation of brain extracts and transfer to nitrocellulose membranes permitted the quantitation of the protein and the study of the carbohydrate chains of tissue-bound transferrins by biotinylated lectins. An unexpected result in the rabbit was the dense immunocytochemical reaction product in Bergmann glia and Golgi epithelial cells. Reaction in the cytoplasm of oligodendrocytes was relatively faint in this species except for some selected white matter tracts, e.g. the inferior cerebellar peduncles. In sections of rat brain, oligodendrocytes and vessel walls reacted vigorously in all locations. Transferrin levels in rat brain were substantially higher than in rabbit brain. In the rabbit, maximum transferrin levels occurred in the cerebellum.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Transferrin uptake by bone marrow macrophages is independent of the degree of iron saturation.

The uptake of transferrin by macrophages was studied in relation to the degree of iron saturation. Rat bone marrow derived macrophages were incubated with transferrin labelled with 59Fe and 3H. At 37 degrees C the amount of 59Fe incorporated by macrophages was dependent on the time of incubation. 3H labelled transferrin was found degraded in the supernatants of the cell culture (material not precipitated by trichloroacetic acid) in a time dependent fashion. Taking into account the specific activity of 59Fe-3H labelled transferrin, we found that 95% of the transferrin uptake was degraded. This suggests that most of the uptake of transferrin was not mediated by a receptor-dependent mechanism, but by a phase fluid endocytosis. 3H-labelled apotransferrin appears in the supernatant of the cell culture at the same rate as 59Fe-3H labelled diferric transferrin, showing an identical uptake for the two types of transferrin. Uptake of apo- or diferric transferrin by macrophages was identical in relation to time of incubation and the amount of transferrin used. These studies suggest that most of the transferrin uptake by bone marrow macrophages (non-activated or non-elicited cells) is mediated by a non-receptor mechanism that is independent of the degree of transferrin saturation.

Animals↗
PubMed1985

The role of transferrin receptors and iron delivery in mouse embryonic morphogenesis.

The iron-carrying serum protein transferrin is required for the proliferation and differentiation of embryonic tissues in culture. We studied the expression and role of transferrin receptors in two model systems using a monoclonal antibody against the transferrin receptor of mice. The addition of 20-100 micrograms/ml antibody to a chemically defined culture medium containing transferrin (10 micrograms/ml) inhibited morphogenesis and cell proliferation in kidneys and teeth. However, the antibody did not inhibit development when iron was delivered to the cells by a lipophilic iron chelator i.e., by-passing the receptor-mediated pathway. Hence, the binding of the receptor antibody to the receptor apparently did not affect cell proliferation, and the antibody was not toxic to the tissues. Our results suggest that the antibody to the transferrin receptor inhibits development by blocking the normal endocytotic route of iron delivery. Cells derived from embryonic kidneys and teeth expressed the transferrin receptor when cultured as monolayers. However, using immunofluorescent techniques, we were unable to detect the receptor in frozen tissue sections. It is possible that the seeding of cells in monolayer cultures affects the expression of the transferrin receptor, since it is known that all types of cells require transferrin for continued proliferation in culture. Organ-cultured kidney mesenchymal cells are not initially responsive to transferrin, but they acquire responsiveness as a consequence of an inductive tissue interaction. Although it remains unknown as to whether the acquisition of transferrin responsiveness is directly related to the expression of transferrin receptors, our results suggest that transferrin and its receptors play a role in embryonic morphogenesis.

Animals↗
PubMed1999

Binding and utilization of human transferrin by Prevotella nigrescens.

To survive and multiply within their hosts, pathogens must possess efficient iron-scavenging mechanisms. In the present study, we investigate the capacity of Prevotella nigrescens and Prevotella intermedia to use various sources of iron for growth and characterize the transferrin-binding activity of P. nigrescens. Iron-saturated human transferrin and lactoferrin, but not ferric chloride and the iron-free form of transferrin, could be used as sources of iron by P. nigrescens and P. intermedia. Neither siderophore activity nor ferric reductase activity could be detected in P. nigrescens and P. intermedia. However, both species showed transferrin-binding activity as well as the capacity to proteolytically cleave transferrin. To various extents, all strains of P. nigrescens and P. intermedia tested demonstrated transferrin-binding activity. The activity was heat and protease sensitive. The capacity of P. nigrescens to bind transferrin was decreased when cells were grown in the presence of hemin. Preincubation of bacterial cells with hemin, hemoglobin, lactoferrin, fibrinogen, immunoglobulin G, or laminin did not affect transferrin-binding activity. The transferrin-binding protein could be extracted from the cell surface of P. nigrescens by treatment with a zwitterionic detergent. Subjecting the cell surface extract to affinity chromatography on an agarose-transferrin column revealed that it contained a protein having an estimated molecular mass of 37 kDa and possessing transferrin-binding activity. The transferrin-binding activity of P. nigrescens and P. intermedia may permit the bacteria to obtain iron for survival and growth in periodontal pockets.

Culture Media↗
PubMed1984

Uptake and metabolism of transferrin and albumin by rat yolk sac placenta.

The uptake of radiolabeled albumin and transferrin by the rat yolk sac and their subsequent transport to the embryo were studied. Transferrin uptake increases with incubation time whether the results are expressed in terms of the total amount accumulated or per milligram embryo or yolk sac protein, whereas albumin levels increase only in absolute terms. The fate of transferrin and albumin was examined by partitioning 125I into low- and high-molecular-weight fractions. Nearly all the embryonic radioiodine originally derived from transferrin is in the low-molecular-weight fraction, compared with only 60% albumin. These results have been extended by examining the uptake and hydrolysis of transferrin and albumin by the isolated yolk sac. Transferrin is taken up more rapidly than albumin. The release of hydrolyzed transferrin to the incubation medium occurred 40 min after the initiation of incubation, compared with 20 min for albumin. Transferrin uptake by the yolk sac at different transferrin concentrations showed an initial rapid phase followed by a slower linear phase, whereas albumin uptake increased linearly with concentration. There was no competition between the two proteins for uptake. Transferrin was released from the yolk sacs at approximately twice the rate of albumin. Results demonstrate at least two uptake mechanisms in the rat visceral yolk sac, one for transferrin, which probably involves receptor-mediated endocytosis, and one for albumin, by which transferrin can also be transported, which probably involves pinocytotic mechanisms.

Animals↗
PubMed1989

Regulation of Sertoli cell differentiated function: testicular transferrin and androgen-binding protein expression.

The regulation of Sertoli cell function was investigated through an examination of the effects of various hormones, regulatory agents, and culture conditions on testicular transferrin and androgen-binding protein (ABP) synthesis and steady state levels of mRNA. FSH stimulated both transferrin and ABP production 2-fold above control levels. Interestingly, FSH had a differential effect on transferrin and ABP mRNA levels, with 1.25- and 2.0-fold respective increases in steady state levels of mRNA. Insulin and retinol stimulated both transferrin and ABP synthesis in a similar manner. Testosterone had no significant effect on either transferrin or ABP mRNA levels or synthesis. Maximum stimulation of both transferrin and ABP production occurred when Sertoli cell cultures were treated with a combination of FSH, insulin, and retinol, which resulted in a greater than 4-fold stimulation of synthesis and 2-fold stimulation of gene expression. Optimal transferrin and ABP secretion occurred between days 4-6 of Sertoli cell culture and subsequently declined. Sertoli cell number decreased with time in culture, such that approximately a 50% loss of cells was observed after 10 days of culture. The responsiveness of Sertoli cells to regulatory agents was altered by cell density, with a maximum responsiveness achieved at a density of 12 micrograms DNA/2 cm2 for both transferrin and ABP. As the cell density deviated from this level the responsiveness of cells to regulatory agents decreased and approached control values. These observations indicate that the culture conditions and the method of data normalization are important parameters in an analysis of the hormonal regulation of Sertoli cell function. FSH actions on Sertoli cells increased both cellular and excreted cAMP levels but had no effect on cGMP levels. (Bu)2 cAMP affected transferrin and ABP mRNA levels and synthesis in a similar manner, with approximately a 3-fold increase in synthesis and a 1.5-fold increase in steady state levels of mRNA. The minimum and maximum effective concentrations of (Bu)2AMP for both proteins were 1 and 10 microM, respectively. Observations imply that regulatory agents that act via a cAMP-mediated signal transduction mechanism, such as FSH, will probably have similar actions on transferrin and ABP production. In addition, data obtained with insulin and retinol indicate that transferrin and ABP production can be similarly regulated with cAMP-independent signal transduction mechanisms. Results indicate that transferrin and ABP mRNA levels and synthesis are regulated in a coordinate manner with the regulatory agents and culture conditions evaluated.(ABSTRACT TRUNCATED AT 400 WORDS)

Androgen-Binding Protein↗
PubMedDate not supplied

Transferrin saturation, dietary iron intake, and risk of cancer.

PURPOSE: Transferrin saturation of more than 60% has been identified as a cancer risk factor. It is unclear whether dietary iron intake increases the risk of cancer among individuals with transferrin saturation of less than 60%. The purpose of this study was to examine the association of dietary iron intake and the risk of cancer among adults with increased transferrin saturation. METHODS: Analysis of a cohort study, the National Health and Nutrition Examination Survey I Epidemiologic Follow-Up Study, was performed. US adults (aged 25 to 74 years at baseline) were followed up from baseline in 1971-1974 to 1992 (N = 6,309). RESULTS: A total of 7.3% of the US population had a serum transferrin saturation of more than 45% at baseline. Intake of dietary iron was essentially uncorrelated with transferrin saturation (r = 0.04). Compared with individuals who had normal serum transferrin saturation and low dietary iron intake, individuals whose serum transferrin saturation was more than 45% and who had high dietary iron intake also had an increased adjusted relative risk of cancer (2.24; 95% confidence interval [CI], 1.02-4.89). Increased risk was not found for individuals with a transferrin saturation of more than 45% but a normal dietary iron intake (hazard ratio, 1.02; 95% CI, 0.69-1.49). Transferrin saturation levels could be set as low as 41%, and the individuals with high transferrin saturation and high dietary iron intake would still have an increased adjusted relative risk of cancer (hazard ratio, 2.00; 95% CI, 1.04-3.82). CONCLUSIONS: Among persons with increased transferrin saturation, a daily intake of dietary iron more than 18 mg is associated with an increased risk of cancer. Future research might focus on the benefits of dietary changes in those individuals with increased serum transferrin saturation.

Adult↗
PubMed1998

Prevalence of elevated serum transferrin saturation in adults in the United States.

BACKGROUND: A population-based hemochromatosis screening program that uses serum transferrin saturation has been proposed, but few data exist on the number of U.S. adults that such a program would identify for further testing. OBJECTIVE: To determine the prevalence of an initially elevated serum transferrin saturation and the prevalence of concurrently elevated serum transferrin saturation and serum ferritin levels in the adult population of the United States. DESIGN: Nationally representative cross-sectional survey of the noninstitutionalized U.S. civilian population. PARTICIPANTS: 15 839 men and nonpregnant women 20 years of age and older who were examined in the third National Health and Nutrition Examination Survey (1988-1994). MEASUREMENTS: Single measurements of serum transferrin saturations and serum ferritin levels. Cut-off values used to define elevated serum transferrin saturation ranged from greater than 45% to greater than 62%. RESULTS: The prevalence of initially elevated serum transferrin saturation ranged from 1% to 6%. Approximately 11% to 22% of those with elevated serum transferrin saturation had concurrently elevated serum ferritin levels. The prevalence of elevated serum transferrin saturation was lower in women than in men when the same cut-off value was used to define elevated serum transferrin saturation. The prevalence of elevated serum transferrin saturation in non-Hispanic black persons and Mexican-Americans was similar to or slightly less than that in non-Hispanic white persons. The prevalence of elevated serum transferrin saturation in persons 20 to 49 years of age was as high as or higher than that in older adults. CONCLUSIONS: A hemochromatosis screening program that uses a cut-off value of greater than 60% to define elevated serum transferrin saturation would identify an estimated 1.4 to 2.5 million U.S. adults for further testing.

Adult↗
PubMed1992

Identification and isolation of the Leishmania transferrin receptor.

In a previous report, we have presented several lines of evidence, derived from widely different methodologies, suggesting that Leishmania has specific receptors for transferrin with a Kd similar to the mammalian transferrin receptor. This paper describes the identification, purification, and biochemical characterization of Leishmania transferrin receptor. The Leishmania transferrin receptor, detected on intact parasites by immunoperoxidase staining, was first identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by Western blot analysis, using 125I-transferrin, as a 70-kDa protein. It has been isolated initially from Leishmania infantum promastigotes using affinity chromatography on a transferrin-Sepharose column and, subsequently, from Leishmania major promastigotes. The use of polyclonal antisera to the purified 70-kDa Leishmania transferrin receptor and to the purified rat transferrin receptor showed that the two receptors are antigenically distinct. The 70-kDa Leishmania transferrin receptor was subsequently characterized as an integral membrane glycoprotein. The monomeric state of the Leishmania transferrin receptor was demonstrated by gel filtration of purified receptor complexed with 125I-transferrin. Thus, the Leishmania transferrin receptor, unlike the mammalian receptor, is not a disulfide-linked dimer but a single 70-kDa polypeptide.

Animals↗
PubMed1989

Transferrin gene expression and synthesis by cultured choroid plexus epithelial cells. Regulation by serotonin and cyclic adenosine 3',5'-monophosphate.

Primary cultures of rat choroid plexus epithelial cells were established and used to investigate the role of the choroid plexus in the synthesis and secretion of transferrin. Transferrin gene expression was determined by a Northern blot analysis with a transferrin cRNA probe. A single transferrin mRNA species was detected and found to be the same size as the transcripts in the liver and Sertoli cells. Immunoprecipitation of radiolabeled secreted proteins with an antiserum transferrin antibody demonstrated that cultured choroid plexus epithelial cells synthesize and secrete a 70-kDa species of transferrin. Levels of transferrin secretion by rat choroid plexus epithelial cells in culture were measured by radioimmunoassay. Treatment of the choroid plexus epithelial cells in culture with cell-permeable cAMP analogs or serotonin led to time- and concentration-dependent changes in the levels of transferrin in the medium. Dibutyryl-cAMP and 8-bromo-cAMP decreased the levels of transferrin synthesized and secreted by choroid plexus epithelial cells with an EC50 value of 30 nM. Serotonin, however, increased the levels of transferrin with an EC50 value of 100 nM. A concomitant change in transferrin mRNA concentrations was observed in response to serotonin. These data suggest that the synthesis of transferrin by the choroid plexus is reciprocally regulated by the neurotransmitter serotonin and by regulatory agents coupled to adenylate cyclase. Regulatory agents such as serotonin may have a critical role in modulating the proteins synthesized by the choroid plexus, thereby influencing the composition of the cerebrospinal fluid.

Animals↗
PubMed1986

Insulin stimulates cellular iron uptake and causes the redistribution of intracellular transferrin receptors to the plasma membrane.

Insulin stimulates the accumulation of iron by isolated fat cells by increasing the uptake of diferric transferrin. Analysis of the cell-surface binding of diferric 125I-transferrin indicated that insulin caused a 3-fold increase in the cell surface number of transferrin receptors. This result was confirmed by the demonstration that insulin increases the binding of an anti-rat transferrin receptor monoclonal antibody (OX-26) to the surface of fat cells. The basis of this effect of insulin was examined by investigating the number of transferrin receptors in membrane fractions isolated from disrupted fat cells. Two methods were employed. First the binding isotherm of diferric 125I-transferrin to the isolated membranes was studied. Second, the membranes were solubilized with detergent, and the number of transferrin receptors was measured by immunoblotting using the monoclonal antibody OX-26. It was observed that insulin treatment of intact fat cells resulted in an increase in the number of transferrin receptors located in the isolated plasma membrane fraction of the disrupted fat cells. Furthermore, the increase in the number of plasma membrane transferrin receptors was associated with a concomitant decrease in the transferrin receptor number in a low density microsome fraction previously shown to consist of intracellular membranes. This redistribution of transferrin receptors between cellular membrane fractions in response to insulin is remarkably similar to the regulation by insulin of glucose transporters and type II insulin-like growth factor receptors. We conclude that insulin stimulates fat cell iron uptake by a mechanism that may involve the redistribution of transferrin receptors from an internal membrane compartment (low density microsomes) to the cell surface (plasma membrane).

Adipose Tissue↗
PubMed1987

Insulin elicits a redistribution of transferrin receptors in 3T3-L1 adipocytes through an increase in the rate constant for receptor externalization.

Incubation of 3T3-L1 adipocytes with insulin at 37 degrees C resulted in a 2-fold increase in specific binding of transferrin to cell-surface receptors, as measured by a subsequent incubation of cells at 4 degrees C with 125I-transferrin. The insulin concentration required for half-maximal effect was 10 nM, and the half-time for insulin action was 40 s. By comparison, insulin stimulated hexose transport in 3T3-L1 adipocytes with a half-maximal effect at 8 nM and a half-time of 105 s. Scatchard analysis of 125I-transferrin binding to cells at 4 degrees C showed that the insulin-induced increase in transferrin receptor binding was due to an increase in the number of surface transferrin receptors. When cells were incubated for 2 h at 37 degrees C with 125I-transferrin to achieve steady-state binding and then exposed to insulin, there was a 1.7-fold increase in surface-bound transferrin (acid-sensitive) and a corresponding decrease in intracellularly bound transferrin (acid-insensitive). Thus, insulin elicits translocation of intracellular transferrin receptors to the plasma membrane. Concomitant with the 2-fold increase in surface receptors in response to insulin, there was a 2-fold increase in the rate of 59Fe3+ uptake from 59Fe3+-loaded transferrin. The rate of externalization of the intracellular 125I-transferrin-receptor complex at 37 degrees C was determined for basal and insulin-treated cells. Insulin increased the first-order rate constant for this process 1.7-fold. The effect of insulin on the rate of externalization is sufficient to account for the increase in surface transferrin receptors.

Adipose Tissue↗