Biomedical subjects

D B Donner

Publications and source records attributed to D B Donner.

At least 37 records · Page 2Linked to original sources

PubMed1996

Inhibition of tumor necrosis factor signal transduction in endothelial cells by dimethylaminopurine.

Tumor necrosis factor (TNF) promotes diverse responses in endothelial cells that are important to the host response to infections and malignancies; however, less is known of the postreceptor events important to TNF action in endothelial cells than in many other cell types. Since phosphorylation cascades are implicated in cytokine signaling, the effects of the protein kinase inhibitor dimethylaminopurine (DMAP) on TNF action in bovine aortic endothelial cells (BAEC) were investigated. In BAEC, TNF promotes phosphorylation of eukaryotic initiation factor 4E (eIF-4E), c-Jun N-terminal kinase (JNK) and ceramide-activated protein kinase activities, Jun-b expression, prostacyclin production, and, when protein synthesis is inhibited, cytotoxicity. DMAP abrogated or significantly attenuated each of these responses to TNF, without affecting the specific binding of TNF to its receptors. Histamine, another agent active in the endothelium, promotes phosphorylation of elongation factor-2 (EF-2) and prostacyclin production, but not phosphorylation of eIF-4E in BAEC. Histamine-stimulated EF-2 phosphorylation was not inhibited and prostacyclin production was unaffected by DMAP. These observations demonstrate that a distinct signal transduction cascade, which can be selectively inhibited by DMAP, promotes the response of BAEC to TNF. Thus, we have identified a reagent, DMAP, that may be useful for characterizing the TNF signal transduction pathway.

Adenine↗

PubMed1996

Tumor necrosis factor promotes phosphorylation and binding of insulin receptor substrate 1 to phosphatidylinositol 3-kinase in 3T3-L1 adipocytes.

Chronic incubation of 3T3-L1 adipocytes with tumor necrosis factor (TNF) induces a state of insulin resistance characterized by a diminished ability of insulin to induce phosphorylation of the beta subunit of its own receptor and insulin receptor substrate 1 (IRS-1). When adipocytes are briefly pretreated with TNF and then stimulated with insulin, tyrosine phosphorylation of IRS-1 increases above the level induced by insulin alone. By itself, TNF induces the time-dependent tyrosine phosphorylation of proteins in 3T3-L1 adipocytes. Among these is IRS-1, a docking protein with tyrosine phosphorylation sites that bind cytoplasmic signaling molecules that contain Src homology 2 (SH2) domains. TNF stimulation of 3T3-L1 adipocytes also promotes the association of the p85 regulatory subunit of phosphatidylinositol 3-kinase (PI 3-kinase) with IRS-1 and also its tyrosine phosphorylation. In murine 3T3-L1 adipocytes, IRS-1 and PI 3-kinase phosphorylation and the association of these proteins are promoted by murine TNF, which interacts with the type 1 and type 2 TNF receptors. Human TNF, which binds to the murine type 1 TNF receptor selectively, also promotes IRS-1 phosphorylation and binding of IRS-1 to PI 3-kinase. This is the first demonstration that a member of the TNF/nerve growth factor receptor superfamily can use an IRS-1 signaling system as a component of its cellular response and provides a mechanism through which TNF receptors may engage downstream elements in signaling pathways.

Adipocytes↗

PubMed1996

Macrophage-stimulating protein, a ligand for the RON receptor protein tyrosine kinase, suppresses myeloid progenitor cell proliferation and synergizes with vascular endothelial cell growth factor and members of the chemokine family.

Macrophage-stimulating protein (MSP), originally identified as an inducer of murine resident macrophage responsiveness to chemoattractants, is a ligand for human RON/murine STK receptor protein tyrosine kinases. Since STK was cloned from populations enriched for hematopoietic stem cells, we initiated studies on the effects of MSP on colony formation by granulocyte-macrophage (CFU-GM), erythroid (BFU-E), and multipotential (CFU-GEMM) myeloid progenitor cells. MSP alone had no colony stimulating activity. However, MSP caused about a 50% suppression of CFU-GM colony formation induced by synergistic combinations of SLF or Flt-L plus GM-CSF, G-CSF, or IL-3 and of BFU-E and CFU-GEMM colonies induced by SLF or Flt3-L plus Epo or Epo and IL-3. In contrast, MSP had no effect on progenitors stimulated by one growth factor. MSP also suppressed colony formation by stimulated cord blood progenitors, but only after preinduction to a rapidly cycling state. It was previously reported that several members of the chemokine family synergistically suppress myeloid progenitor proliferation. Likewise, synergistic suppression was observed when MSP was paired with VEGF, MIP-1 alpha, IL-8, PF4, MCP-1, IP-10, or ENA-78, or when VEGF was paired with the chemokines; and the required MSP concentration was more than 100-fold less than for MSP alone. Additionally, MSP or VEGF inhibited proliferation of the human myeloid growth factor-dependent cell line, M07e, but a sustained effect required multiple additions over time. At the least, some of the MSP suppressive effects on myeloid progenitors, as assessed on single isolated CD34 marrow cells, appeared to be directly on the progenitors; sustained additions of MSP were required to see this effect. The suppressive action of MSP and its synergism with proteins of the chemokine family may be of relevance to regulation of blood cell production.

Bone Marrow Cells↗

PubMed1995

Association of a RING finger protein with the cytoplasmic domain of the human type-2 tumour necrosis factor receptor.

A human gene encoding a protein that specifically binds to the intracellular domain of the 75 kDa type-2 tumour necrosis factor (TNF) receptor (TNFR-2IC) has been identified using the yeast-based two-hybrid system. The N-terminal half of the TNF receptor-associated protein (TRAP) contains RING finger and zinc finger motifs often found in DNA-binding proteins including transcription factors. The 2.4 kb TRAP mRNA was barely detectable, if present at all, in lung, and variably expressed in heart, liver, placenta, brain, skeletal muscle, kidney and the pancreas; interestingly, the TRAP was more highly expressed in transformed cell lines than in normal tissues. This observation may be consistent with a role for this TRAP in promoting or regulating cellular proliferation. After in vitro transcription/translation and 35S labelling the TRAP was precipitated using a fusion protein consisting of glutathione S-transferase and the intracellular domain of TNFR-2 (TNFR-2IC), which showed that the two proteins directly interact in a mammalian cell-free system and also that identification of the TRAP was not an artifact of the two-hybrid system. By using truncated TNFR-2ICs for in vitro precipitation of 35S-TRAP, it was shown that the C-terminal half of the TNFR-2IC contains the domain necessary for interaction with TRAP. The TRAP identified in the present study shares considerable homology with, and may be the human homologue of, a mouse protein, TNF receptor-associated factor 2 (TRAF2), that binds mouse TNFR-2.

Amino Acid Sequence↗

PubMed1995

Vascular endothelial cell growth factor promotes tyrosine phosphorylation of mediators of signal transduction that contain SH2 domains. Association with endothelial cell proliferation.

Vascular endothelial cell growth factor (VEGF), an endothelial cell-specific mitogen that plays an important role in angiogenesis, promotes the tyrosine phosphorylation of at least 11 proteins in bovine aortic endothelial cells (BAEC). Proteins immunoprecipitated from lysates of control- and VEGF-stimulated BAEC with antisera to phospholipase C-gamma (PLC-gamma) were fractionated by SDS-polyacrylamide gel electrophoresis and transferred to Immobilon-P. Evaluation of the Western blots with antisera to phosphotyrosine demonstrated that PLC-gamma and two proteins (100 and 85 kDa) that associate with PLC-gamma were phosphorylated in response to VEGF. By using antisera specific to other mediators of signal transduction that contain SH2 domains for immunoprecipitation, it was demonstrated that VEGF promotes phosphorylation of phosphatidylinositol 3-kinase, Ras GTPase activating protein (GAP), and the oncogenic adaptor protein NcK. Proteins of M(r) consistent with the VEGF receptors Flt-1 and Flk-1/KDR were also tyrosine phosphorylated in stimulated cells. Tyrosine-phosphorylated Nck, PLC-gamma, and two GAP-associated proteins, p190 and p62, were in GAP immunoprecipitates of VEGF-stimulated BAEC, and tyrosine-phosphorylated NcK was in phosphatidylinositol 3-kinase immunoprecipitates. These observations suggest that VEGF promotes formation of multimeric aggregates of VEGF receptors with proteins that contain SH2 domains and activate various signaling pathways. VEGF-promoted proliferation of endothelial cells and tyrosine phosphorylation of SH2 domain containing signaling molecules were inhibited by the tyrosine kinase inhibitor genistein.

Adaptor Proteins, Signal Transducing↗

PubMed1995

Identification of a protein with homology to hsp90 that binds the type 1 tumor necrosis factor receptor.

The yeast-based two hybrid has been used to identify a novel protein that binds to the intracellular domain of the type 1 receptor for tumor necrosis factor (TNFR-1IC). The TNF receptor-associated protein, TRAP-1, shows strong homology to members of the 90-kDa family of heat shock proteins. After in vitro transcription/translation and 35S labeling, TRAP-1 was precipitated using a fusion protein consisting of glutathione S-transferase and TNFR-1IC, showing that the two proteins directly interact. The ability of deletion mutants of TNFR-1 to interact with TRAP-1 was tested using the two hybrid system. This showed that the amino acid sequences that mediate binding are diffusely distributed outside of the domain in the C terminus of TNFR-1IC that signals cytotoxicity. The 2.4-kilobase TRAP-1 mRNA was variably expressed in skeletal muscle, liver, heart, brain, kidney, pancreas, lung, and placenta. TRAP-1 mRNA was also detected in each of eight different transformed cell lines. Identification of TRAP-1 may be an important step toward defining how TNFR-1, which does not contain protein tyrosine kinase activity, transmits its message to signal transduction pathways.

Amino Acid Sequence↗

PubMed1995

Myeloid progenitor cell regulatory effects of vascular endothelial cell growth factor.

Vascular endothelial cell growth factor (VEGF) is a ligand for the tyrosine kinase receptor Flk-1/KDR and Flt1 and is considered to be an endothelial cell specific mitogen that plays an important role in angiogenesis. Since Flk-1 mRNA has been detected in primitive and more mature hematopoietic cells, recombinant human VEGF was evaluated for its influence on hematopoiesis, which was assayed as in vitro colony formation by myeloid progenitor cells from human bone marrow. VEGF enhanced colony formation by mature subsets of granulocyte-macrophage and erythroid progenitor cells that had been stimulated with a colony stimulating factor. In contrast, VEGF inhibited colony formation by more immature subsets of granulocyte-macrophage, erythroid and multipotential progenitor cells synergistically stimulated to proliferate with a colony stimulating factor and either steel factor or the ligand for the Flt-3 receptor tyrosine kinase. VEGF produced effects similar to those given above on purified CD34 progenitor cells from bone marrow and VEGF effects were neutralized by VEGF antibodies. However, when assessed for effects on single sorted CD34 cells, VEGF only enhanced or suppressed colony formation by granulocyte-macrophage progenitor cells and the amplitude of the response was less than that observed when populations of these cells were tested. In the single cell assays, VEGF had no effect on colony formation by erythroid or multipotential progenitors. These results suggest that the effects of VEGF, which were not species specific, are mediated by both direct and indirect actions on the progenitors and thereby identify new activities for this important factor.

Animals↗

PubMed1994

Aggregation of the intracellular domain of the type 1 tumor necrosis factor receptor defined by the two-hybrid system.

The yeast-based two hybrid system has been used to determine whether oligomerization of the intracellular domain of the 55-kDa type 1 tumor necrosis factor (TNF) receptor may occur during TNF action. This assay depends upon reconstitution of the function of the GAL4 transcriptional activator through interaction of a protein fused to the GAL4 DNA binding domain with a protein fused to the transcriptional activation domain of GAL4. Fusion of the type 1 TNF receptor intracellular domain with the DNA binding domain and the transactivation domain of GAL4 led to activation of the lacZ indicator gene, demonstrating interaction of the receptor intracellular domain with itself. A HeLa cell cDNA library was searched for proteins that interact with the intracellular domain of the type 1 TNF receptor. A protein corresponding to amino acids 329-426 in the type 1 TNF receptor intracellular domain was identified by this screen. The aggregation domain was further defined by testing the ability of deletion mutants of the type 1 TNF receptor intracellular region to interact with the complete intracellular domain. These experiments map the aggregation domain to a sequence of amino acids previously shown to be responsible for mediating TNF-induced cytotoxicity. These results suggest that aggregation of type 1 TNF receptor intracellular domains may be important in TNF signal transduction.

Cloning, Molecular↗

PubMed1994

Acute metabolic effects of human recombinant tumor necrosis factor beta in the rat.

BACKGROUND: Cancer cachexia is associated with several alterations in host metabolism, including hypoaminoacidemia and an increase in gluconeogenesis (GLC) and lipolysis. Tumor necrosis factor beta (TNF beta), a lymphokine released by mitogen-activated T lymphocytes and several cancer cell lines, causes an increase in lipolysis in 3T3L1 adipocytes. Since little is known about the metabolic effects of TNF beta in vivo, we examined its acute effects in the rat. METHODS: Twenty-eight male Fischer rats were injected intraperitoneally with TNF beta (250 micrograms/kg) or saline (CTL), and after 4 h, isolated hepatocytes were obtained (by in situ collagenase liver perfusion [n = 12]) or aortic blood was collected (n = 16). Hepatocytes were incubated with 10 mM alanine (ALA) or 10 mM lactate (LAC), and glucose production was measured. Rates of GLC (nmol glucose/10(6) cells/min) were determined by linear regression. Plasma lactate, glucose, insulin, and amino acids (AA) (nmol/ml) were measured, and values were expressed as means +/- SEM. Comparisons between groups were made by unpaired t test or Mann-Whitney U test, and significance was defined as p < 0.05. RESULTS: TNF beta caused a 130% increase in gluconeogenesis from alanine (2.7 +/- 0.5 vs 1.2 +/- 0.2 nmol glucose/10(6) cells/min, TNF vs CTL), and a 60% increase from lactate (7.5 +/- 1.0 vs 4.6 +/- 0.5 nmol glucose/10(6) cells/min, TNF vs CTL). Plasma insulin levels in TNF treated rats were 1.2 +/- 0.2 ng/ml compared to 1.1 +/- 0.2 ng/ml in CTL. Total amino acid levels in TNF treated rats were 3,175 +/- 111 nmol/ml compared to 3,190 +/- 103 nmol/ml in CTL. CONCLUSION: In vivo TNF beta causes an increase in hepatic gluconeogenesis from alanine and lactate with no change in plasma insulin or amino acids.

Amino Acids↗

PubMed1993

Human insulin receptor mutated at threonine 1336 functions normally in Chinese hamster ovary cells.

Phosphorylation of threonine 1336 of the human insulin receptor (HIR) is stimulated by insulin or 4 beta-phorbol 12-myristate 13-acetate in Chinese hamster ovary (CHO) transfectant cells expressing the wild type receptor (CHO/HIR). To examine the role of this phosphorylation in insulin signal transduction, a mutant human insulin receptor, in which threonine 1336 was replaced with asparagine, has been stably expressed in CHO cells (CHO/HIRT1336N). CHO cell lines expressing equivalent numbers of the wild type or the mutant receptor were developed, which bound 125I-insulin comparably (Kd = 0.1 nM). After stimulation of CHO/HIR or CHO/HIRT1336N cells with insulin, the wild type and mutant receptors internalized the hormone and were down-regulated with similar rates. Hormone stimulation of the receptor tyrosine kinase activity was also unaffected by the mutation. Metabolic and mitotic effects of insulin were also unimpaired by the mutation. Thus, insulin stimulated phosphatidylinositol 3-kinase activity, glycogen synthesis, and thymidine incorporation into DNA similarly in CHO/HIR and CHO/HIRT1336N cells. These data suggest that by itself phosphorylation of threonine 1336 has no significant effect on insulin binding, regulation of insulin receptor expression, or insulin signal transduction.

Animals↗

PubMed1993

Identification of a new receptor subtype for tumor necrosis factor-alpha.

Two distinct receptors, which bind both tumor necrosis factor-alpha and tumor necrosis factor-beta (TNF-alpha and TNF-beta), have been previously identified and cloned from transformed cells. The present study identifies a novel receptor subtype in normal human liver which binds TNF-alpha but not TNF-beta. TNF-alpha but not TNF-beta competes for 125I-TNF-alpha binding and incorporation into affinity-labeled complexes in human liver plasma membranes (HLPM). Antisera to the cloned receptors competed for 125I-TNF-alpha binding to plasma membranes isolated from various transformed cell lines but not to HLPM. However, mRNAs corresponding in size to both known TNF receptors were detected in liver RNA, making it likely that post-transcriptional modifications account for the TNF-alpha specificity of HLPM. These observations suggest that the effects of TNF-alpha and TNF-beta on some normal tissues may be more distinct than previously realized.

Antibodies, Monoclonal↗

PubMed1993

Interaction of the human insulin receptor tyrosine kinase from the baculovirus expression system with protein kinase C in a cell-free system.

The cytoplasmic domain of the human insulin receptor (HIR) from the baculovirus expression system (BIRK) is a soluble, constitutively activated protein-tyrosine kinase. In a cell-free system, BIRK is phosphorylated on serine and threonine residues by protein kinase C (PKC) purified from rat brain. Two-dimensional tryptic phosphopeptide mapping of PKC-phosphorylated BIRK identified one phosphothreonine and three phosphoserine peptides, which were also in tryptic digests of insulin receptors from insulin- or PMA-treated Chinese hamster ovary (CHO) cells transfected with the HIR. After Lys-C proteolysis of PKC-phosphorylated BIRK, radioactive phosphopeptides were purified on a C8 reverse-phase high pressure liquid chromatography column. Amino acid sequence analysis identified a phosphothreonine peptide corresponding to amino acids 1331-1340 of the HIR. This peptide contains only one threonine, amino acid 1336, which is identified as a site for PKC phosphorylation in BIRK. CHO cells transfected with the wild type (CHO/HIR) or a mutant human insulin receptor (CHO/HIRT1336N), in which threonine 1336 was substituted with asparagine, were 32P labeled and then stimulated with insulin or phorbol 12-myristate 13-acetate (PMA). Two-dimensional phosphopeptide analysis of the HIR revealed that phosphorylation of phosphothreonine peptide T, shown to be in PKC-phosphorylated BIRK, was increased by insulin or PMA. However, the corresponding peptide was not in the mutant receptor. Therefore, the present study directly identifies threonine 1336 in the HIR as a phosphorylation site for insulin and PMA. These data also show that BIRK can be used as a model for the study of the regulation of the insulin receptor kinase.

Animals↗

PubMed1993

Tumour necrosis factor-induced cytotoxicity is accompanied by intracellular mitogenic signals in ME-180 human cervical carcinoma cells.

Tumour necrosis factor-alpha (TNF) induced a cytotoxic response in ME-180 human cervical carcinoma cells in vitro. This cytotoxic response was accompanied by a temporal series of intracellular signals that are commonly triggered by a mitogenic stimulus: increased c-fos (20-30 min) and c-myc (40-60 min) expression, increased activity of ornithine decarboxylase (3 h), increased intracellular polyamine content (7 h) and increased thymidine incorporation into DNA (14 h). A cytotoxic response independent of these mitogenic signals could not be explained by an induction of interleukin-6, which is an autocrine cytotoxic agent in some cell types; nor by a biphasic, dose-dependent response in which low concentrations of TNF are mitogenic and higher concentrations are cytotoxic. Conversely, a dependent role of these mitogenic signals was suggested by the absence of a TNF-promoted increase in thymidine incorporation into DNA in an ME-180 clone that is resistant to TNF-induced cytotoxicity. A decrease in the proliferation rate of TNF-sensitive cells induced by either alpha-difluoromethylornithine treatment (resulting in polyamine depletion) or serum starvation rendered the cells insensitive to TNF-induced cytotoxicity, further suggesting a role for mitogenic signals and cell division in TNF-mediated cytotoxicity. However, inhibiting proliferation with cycloheximide resulted in increased sensitivity to TNF, implying that mitogenesis itself was not essential for a cytotoxic response. TNF induced DNA fragmentation in sensitive cells, suggesting that cytotoxicity occurred via apoptosis.

Apoptosis↗

PubMed1991

Identification and characterization of receptors for tumor necrosis factor-alpha in the brain.

Specific receptors for murine TNF have been identified in homogenates of rodent brain. These receptors are saturable and bind TNF with sufficient affinity to ensure occupancy by cytokine elaborated during infection. 125I-mTNF was detected in four specific complexes of Mr 130,000, 90,000, 66,000 and 60,000 after affinity labeling. Solubilization of brain membranes into detergent increased binding capacity 4-fold which indicates the presence of latent receptors for mTNF in the brain. Specific binding was greatest in the brainstem, least in the cerebellum and was also detected in the cortex, thalamus and basal ganglia.

Animals↗

PubMed1991

Phosphorylation of the proto-oncogene product eukaryotic initiation factor 4E is a common cellular response to tumor necrosis factor.

The initiation of mRNA translation is regulated by the reversible phosphorylation of several initiation factors. We report here that tumor necrosis factor-alpha (TNF) rapidly stimulates phosphorylation of one such factor, an mRNA cap binding protein, in several cell types which are important in vitro models of TNF action. This protein has been purified, sequenced, and identified as the proto-oncogene product eukaryotic initiation factor 4E. These data show that phosphorylation of a key component of the cellular translational machinery is a common early event in the various actions of TNF in diverse cell types.

Amino Acid Sequence↗

PubMed1990

Identification, characterization, and homologous up-regulation of latent (cryptic) receptors for tumor necrosis factor-alpha in rat liver plasma membranes.

A population of latent (cryptic) receptors for tumor necrosis factor-alpha (TNF) has been characterized in the rat liver plasma membrane (PM). 125I-TNF bound to high (Kd = 1.51 +/- 0.35 nM) and low (Kd = 13.58 +/- 1.45 nM) affinity receptors in PM. Solubilization of PM with 1% Triton X-100 prior to incubation with 125I-TNF increased both high affinity (from 0.33 +/- 0.04 to 1.67 +/- 0.05 pmol/mg of protein) and low affinity (from 1.92 +/- 0.16 to 7.57 +/- 0.50 pmol/mg of protein) TNF binding without affecting the affinities for TNF. Digestion of intact PM with chymotrypsin abolished most of the TNF binding capacity of PM. However, substantial binding activity was recovered by solubilization of chymotrypsin-treated PM with 1% Triton X-100, suggesting the presence of a large latent pool of TNF receptors. The affinities of the high and low affinity sites recovered from chymotrypsin-treated membranes were similar to those of intact PM. Affinity labeling of receptors whether from PM, solubilized PM, or membranes digested with chymotrypsin and then solubilized resulted in cross-linking of 125I-TNF into Mr 130,000, 90,000, and 66,000 complexes. Thus, the properties of the latent TNF receptors were similar to those initially accessible to TNF. To determine if exposure of latent receptors is regulated by TNF, 125I-TNF binding to control and TNF-pretreated membranes was assayed. Specific binding was increased by pretreatment with TNF (p less than 0.05), demonstrating that hepatic PM contains latent TNF receptors whose exposure is promoted by TNF. Homologous up-regulation of TNF receptors may, in part, be responsible for sustained hepatic responsiveness during chronic exposure to TNF.

Affinity Labels↗

PubMed1990

The down-regulation of alpha-interferon receptors in human lymphoblastoid cells: relation to cellular responsiveness to the antiproliferative action of alpha-interferon.

Human lymphoblastoid cell lines (Daudi, Daudi subclones, Raji and MOLT-4) were compared for sensitivity to the antiproliferative action of alpha-interferon (IFN-alpha) and down-regulation of IFN-alpha receptors. IFN-sensitive and IFN-resistant cell lines have similar numbers (2-4000/cell) of high affinity (20-75 pM) IFN-alpha receptors. Treatment of IFN-sensitive cells with low concentrations (3-10 pM) of IFN-alpha results in low receptor occupancy and nearly complete (greater than 95%) down-regulation of cell surface IFN-alpha receptors within 5 h. Treatment of resistant cells with higher IFN concentrations (30 pM) only results in partial (approximately 60%) receptor down-regulation that is directly related to receptor occupancy. Receptor-receptor interactions, induced by IFN-alpha binding, may account for the enhanced down-regulation of IFN-alpha receptors in IFN-sensitive cells. Such interactions apparently do not occur in IFN-resistant lymphoblastoid cell lines.

Cell Division↗

PubMed1989

Mediation of anorexia by human recombinant tumor necrosis factor through a peripheral action in the rat.

Human recombinant tumor necrosis factor (TNF) produces significant anorexia in the rat which persists for up to 24 h after a single dose (5 micrograms/325 g rat). Dose-response studies indicate similar potencies for TNF following central or peripheral administration. Brain 125I-TNF levels were more than 100-fold greater after intracerebroventricular than i.v. injection, whereas blood levels of radioactivity were quite similar following both routes of administration. Gel filtration chromatography and precipitation by trichloroacetic acid showed that the radioactive label which exited the central nervous system was associated with intact TNF. The rapid effusion of 125I-TNF from the central nervous system resulted in detection of similar levels of the cytokine in a number of important target tissues (skin, muscle, fat) relative to that detected after peripheral administration. After i.v. or intracerebroventricular administration, blood levels of TNF declined rapidly to nearly undetectable levels over 4 h. However, the anorexia induced by TNF was sustained, and feeding remained depressed between 6 and 24 h postadministration. These observations suggest that TNF produces its anorectic effects at peripheral sites, possibly through mediators.

Animals↗