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PubMed1994

Adipsin/acylation stimulating protein system in human adipocytes: regulation of triacylglycerol synthesis.

Through their capacity to store fatty acids as triacylglycerol molecules, adipocytes serve a vital physiologic role. This study presents further evidence that this process can be modulated in human adipocytes by the adipsin/acylation stimulating protein (ASP) pathway and suggests a novel function for the product of this system--ASP. The data demonstrate the following: (1) ASP stimulates triacylglycerol synthesis within adipocytes, and this occurs to a greater extent in differentiating than undifferentiated cells (242% +/- 32% vs 168% +/- 11%, p < 0.01, respectively, at an ASP concentration of 88 ng/mL; (2) ASP does not affect the Km for triacylglycerol synthesis but does substantially increase Vmax; (3) when ASP is generated in vitro through incubation of its precursor proteins under appropriate conditions, triacylglycerol synthesis increases to the same extent as when plasma-purified ASP is added to the medium; (4) human adipocytes contain mRNA for the specific serine protease adipsin and the two precursor proteins C3 and factor B required to interact for the production of ASP; and (5) the extent to which cultured differentiating adipocytes produce ASP is proportional to the degree to which they have accumulated triacylglycerol mass during differentiation (r2 = 0.7523, p < 0.0005). These findings provide the first evidence for the existence of the adipsin/ASP pathway in human adipocytes, and this may markedly enhance our understanding of the processes which regulate triacylglycerol clearance from plasma.

Acylation↗
PubMed1987

Nucleoprotein complexes that regulate gene expression in adipocyte differentiation: direct participation of c-fos.

Adipocyte differentiation is accompanied by the transcriptional activation of many new genes, including a putative lipid-binding protein termed adipocyte P2 (aP2). The aP2 gene contains a regulatory element (FSE2) 124 bases 5' to its start of transcription. This element binds nuclear factors in sequence-specific and differentiation-dependent fashion as determined by altered mobility in gel retardation assays. Deletion analysis of promoter-linked transfection assays and competition of these constructions in cells with a synthetic FSE2 element suggest that trans-acting factors bind to this region and act as negative regulators of aP2 gene activity in preadipocytes. c-fos appears to participate directly in this nucleoprotein complex, as demonstrated by the ability of antibodies to c-fos to disrupt specific binding of factors to the FSE2 sequence but not to factor-binding sequences from several other genes. Antibodies to c-fos specifically immunoprecipitate protein complexes covalently bound to FSE2 DNA via UV cross-linking.

Adipose Tissue↗
PubMed1980

Partial amino acid sequence of human factor D:homology with serine proteases.

Human factor D purified to homogeneity by a modified procedure was subjected to NH2-terminal amino acid sequence analysis by using a modified automated Beckman sequencer. We identified 48 of the first 57 NH2-terminal amino acids in a single sequencer run, using microgram quantities of factor D. The deduced amino acid sequence represents approximately 25% of the primary structure of factor D. This extended NH2-terminal amino acid sequence of factor D was compared to that of other trypsin-related serine proteases. By visual inspection, strong homologies (33--50% identity) were observed with all the serine proteases included in the comparison. Interestingly, factor D showed a higher degree of homology to serine proteases of pancreatic origin than to those of serum origin.

Amino Acid Sequence↗
PubMed1985

In vitro biosynthesis of complement protein D by U937 cells.

Preliminary studies demonstrating the secretion of antigenic D by blood monocytes/macrophages led us to study the biosynthesis of D by U937 cells, a human monocyte cell line. The kinetics of secretion of D into cell culture supernatants were followed by a solid-phase radioimmunoassay and by hemolytic assay. Daily synthesis of antigenic D was nearly linear (mean +/- 1 SD = 5.3 +/- 2.2 ng D/10(6) cells) over a 6-day period. The D produced after day 2 was hemolytically active, with a specific hemolytic activity greater than (although in the same range as) D in normal serum. Cycloheximide (10(-7) M) inhibited D synthesis, which returned to the levels found in untreated cells after removal of the inhibitor. Supernatants and lysates of cells grown in the presence of [35S]methionine were incubated with rabbit anti-D serum or FD10-1, a monoclonal anti-D antibody, bound to protein A-agarose. Autoradiograms of SDS-PAGE analysis of the precipitates demonstrated a main band of an approximate m.w. of 24,000, co-migrating with purified 125I-D. Identity of this band with D was established by blocking with excess purified D. Pulse-chase studies with the use of [35S]cysteine demonstrated a single D band both intra and extracellularly. Both forms of D had the same apparent m.w. which was approximately 3000 heavier than control 125I-D. These data demonstrate that U937 cells synthesize functionally active D, which appears to be structurally and antigenically similar to D in serum.

Amino Acids↗
PubMed1982

Serum hemolytic factor D values in children with steroid-responsive idiopathic nephrotic syndrome.

Serum hemolytic factor D activity, an important component of the ACP, was measured in patients with SR-INS. The mean serum factor D hemolytic activity of patients with SR-INS in relapse was significantly reduced compared to the mean of the control group. Twenty-one of 27 SR-INS patients in relapse (78%) had reduced serum factor D activity. In contrast, the mean serum factor D hemolytic activity of SR-INS patients in remission was not significantly different from that in the control group. Factor D hemolytic activity was also reduced in other types of renal disease with the nephrotic syndrome. Serum factor D values were highly correlated with the serum albumin concentration. Although hemolytic factor D activity could not be detected in the urine, the low molecular weight and the significant correlation with serum albumin concentration suggest that urinary loss is responsible for the low serum levels of factor D. Deficient serum values of factor D may contribute to the increased susceptibility of SR-INS patients in relapse to bacterial infections with organisms which activate the ACP.

Adolescent↗
PubMed1992

Differentiation-dependent expression of retinoid-binding proteins in BFC-1 beta adipocytes.

Recently, we demonstrated that adipose tissue plays an important role in retinol storage and retinol-binding protein (RBP) synthesis. Our data suggested that RBP expression in adipose tissue is dependent on the state of adipocyte differentiation. To examine this possibility, we explored the differentiation-dependent expression of RBP using BFC-1 beta preadipocytes, which can be stimulated to undergo adipose differentiation. Total RNA was isolated from undifferentiated (preadipocytes) and differentiated (adipocytes) BFC-1 beta cells and analyzed by Northern blotting. RBP mRNA was not detected in the preadipocytes, but considerable RBP mRNA was present in differentiated BFC-1 beta cells. In BFC-1 beta cells, induced to differentiate with insulin and thyroid hormone, RBP mRNA was first detected after 4 days, reached a maximum level by day 10, and remained at this maximum level for at least 2 more days. Cellular retinol-binding protein was expressed at low levels in the BFC-1 beta preadipocytes and the level of expression increased for 6 days after induction to differentiate and slowly declined on later days. Neither the maximum level of RBP expression nor the day on which this level was reached was influenced by the level of retinol provided in the BFC-1 beta culture medium. BFC-1 beta cells secreted newly synthesized RBP into the culture medium at a rate of 43 +/- 14 ng RBP/24 h/10(6) adipocytes. When the BFC-1 beta adipocytes were provided 1.0 microM retinol in the medium, they accumulated the retinol and synthesized retinyl esters. These studies with BFC-1 beta cells confirm that RBP synthesis and secretion and retinol accumulation are intrinsic properties of differentiated adipocytes. Furthermore, they suggest that RBP and cellular retinol-binding protein gene expression are regulated as part of a package of genes which are modulated during adipocyte differentiation.

Adipose Tissue↗
PubMed1985

Biosynthesis of complement protein D by HepG2 cells: a comparison of D produced by HepG2 cells, U937 cells and blood monocytes.

The biosynthesis of complement protein D of the alternative pathway by HepG2 cells, a human hepatocyte cell line, was studied and compared to the biosynthesis of D by U937 cells and blood monocytes. Increasing amounts of antigenic D were detected in HepG2 cell culture supernatants by radioimmunoassay. The kinetics of D synthesis and secretion by HepG2 cells was followed in a pulse-chase study using [35S]cysteine. As analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by autoradiography, only a single D band was seen intra- and extracellularly and both forms had the same apparent molecular weight as D synthesized by U937 cells or purified from serum. Treatment of HepG2 and U937 cells with canavanine, an arginine amino acid analog, to inhibit intracellular processing resulted in slight depression of the apparent molecular weight of D synthesized by these cells. D synthesized by blood monocytes had an apparent molecular weight similar to that synthesized by HepG2 and U937 cells, suggesting that these cell lines do not synthesize and process D differently than normal monocytes. The data demonstrate that the hepatocyte is a site of D synthesis and suggest that D is not synthesized as a precursor molecule.

Canavanine↗
PubMed1987

Adipsin: a circulating serine protease homolog secreted by adipose tissue and sciatic nerve.

Adipsin is a serine protease homolog whose primary structure was predicted from the nucleotide sequence of a differentiation-dependent adipocyte messenger RNA. Immunoblots probed with antisera to synthetic peptides identify two forms of adipsin that are synthesized and secreted by 3T3 adipocytes. These proteins of 44 and 37 kilodaltons are converted to 25.5 kilodaltons by enzymatic deglycosylation. Although adipsin is principally synthesized in adipose tissue, it is also produced by sciatic nerve and is found in the bloodstream. Because of the apparent restriction of adipsin synthesis to tissues highly active in lipid metabolism, its presence in serum, and its modulation in altered metabolic states, this molecule may play a previously unrecognized role in systemic lipid metabolism or energy balance.

Adipose Tissue↗
PubMed1989

Targeting of nonexpressed genes in embryonic stem cells via homologous recombination.

Gene targeting via homologous recombination-mediated disruption in murine embryonic stem (ES) cells has been described for a number of different genes expressed in these cells; it has not been reported for any nonexpressed genes. Pluripotent stem cell lines were isolated with homologously recombined insertions at three different loci: c-fos, which is expressed at a low level in ES cells, and two genes, adipsin and adipocyte P2 (aP2), which are transcribed specifically in adipose cells and are not expressed at detectable levels in ES cells. The frequencies at which homologous recombination events occurred did not correlate with levels of expression of the targeted genes, but did occur at rates comparable to those previously reported for genes that are actively expressed in ES cells. Injection of successfully targeted cells into mouse blastocysts resulted in the formation of chimeric mice. These studies demonstrate the feasibility of altering genes in ES cells that are expressed in a tissue-specific manner in the mouse, in order to study their function at later developmental stages.

Adipose Tissue↗
PubMed1986

Adipocyte P2 gene: developmental expression and homology of 5'-flanking sequences among fat cell-specific genes.

We have isolated the mouse gene encoding adipocyte P2, aP2, the differentiation-dependent adipocyte protein homologous to myelin P2. The aP2 gene is present in a single copy in the mouse and is present in single or few copies in species from human to Drosophila. The entire gene spans 4 kilobases and consists of four exons encoding 25, 57, 34, and 16 amino acids; the overall exon structure is similar to the gene encoding liver fatty acid binding protein. A plasmid vector was constructed containing the entire aP2 gene with flanking sequences, modified by linker insertion. When this gene is stably introduced into 3T3-F442A cells, it is expressed only upon adipose differentiation, with a time course of induction very similar to that of the endogenous aP2 gene. We have compared the DNA sequence of the 5'-flanking region of the aP2 gene to the promoter regions of two other genes activated during adipocyte differentiation, glycerol-3-phosphate dehydrogenase and adipsin, and find a 13-base region of homology (Formula: see text) present in multiple copies in the 5'-flanking region of each gene. An adjacent 15-base sequence is present only in glycerol-3-phosphate dehydrogenase and aP2 genes. Both of these elements share homology with putative viral enhancer core sequences. These results indicate that the aP2 gene contains sequence information necessary for differentiation-dependent expression in fat cells; common elements shared by adipocyte-specific genes may play a role in this process.

Adipose Tissue↗
PubMed1994

Characterization of the surfactant protein A receptor: cell and ligand specificity.

The cell and ligand specificity of the putative surfactant protein A (SP-A) receptor was investigated using a cell receptor assay in which SP-A-coated magnetic beads were incubated with 51Cr-labeled cells at 4 degrees C. Cells that bound to the SP-A-coated beads were isolated by magnetic separation. The recovery of alveolar macrophages was significantly higher than the recovery of peritoneal macrophages, alveolar type II cells, polymorphonuclear leukocytes, COS cells, and U937 cells. In "coincubation" experiments, in which the potential inhibitors were present during the cell recovery assay, SP-A, SP-D, and complement factor 1q (C1q) all inhibited recovery. In contrast, in "preincubation" experiments, in which cells were incubated with potential inhibitors and then washed before the cell recovery assay, only SP-A inhibited cell recovery. Because SP-A binds to SP-D and C1q, we speculate that inhibition of SP-A binding to macrophages by SP-D and C1q in coincubation assays is due to interaction of SP-A with SP-D or C1q, rather than competition for receptor binding. The recovery of alveolar macrophages was also inhibited by preincubation with heat-denatured SP-A, deglycosylated SP-A, and with the collagenase-resistant fragment of SP-A, but not by coincubation with mannan. These results suggest the presence of a receptor on alveolar macrophages with a high degree of specificity for SP-A.

Animals↗
PubMed1990

Sodium butyrate in combination with insulin or dexamethasone can terminally differentiate actively proliferating Swiss 3T3 cells into adipocytes.

Sodium butyrate arrests the growth of actively proliferating Swiss 3T3 cells. A previous report from our laboratory describes the pattern of expression of a representative group of growth-associated genes following treatment of Swiss 3T3 cells with sodium butyrate. The results of this study suggest that sodium butyrate-induced growth arrest involves events which lead to adipocyte differentiation (Toscani, A., Soprano, D.R., and Soprano, K.J. (1988) Oncogene Res. 3, 233-238). However, while sodium butyrate by itself could apparently initiate adipogenesis, it alone was not sufficient to maintain this differentiation state. We now wish to further characterize the role of sodium butyrate in adipocyte differentiation. Subconfluent cultures of Swiss 3T3 cells were treated with sodium butyrate in combination with other agents known to induce Swiss 3T3 cell adipogenesis (e.g. 1-methyl-3-isobutylxanthine, insulin, and dexamethasone) and then analyzed at various times thereafter for: (a) the presence of high concentrations of intracellular lipid as detected by microscopic examination of treated cells following staining with lipid-specific dyes and (b) the expression of four genes known to be modulated during the differentiation of preadipocytes into mature adipocytes (actin, adipsin, lipoprotein lipase, and adipocyte P2). Our results show that sodium butyrate in combination with either insulin or dexamethasone can fully differentiate Swiss 3T3 cells into adipocytes, at least as determined by accumulation of high levels of intracellular lipid. Moreover, the sodium butyrate-mediated process of differentiation can occur in subconfluent, actively proliferating cells. Thus, these experiments describe a new, previously unidentified activity of sodium butyrate and also suggest that this model system may be a useful one to study the relationship between growth arrest and differentiation.

Actins↗
PubMed1987

Isolation of complement protein D from urine of patients with Fanconi's syndrome.

Complement protein D is the least abundant of all complement proteins and, thus, one of the most difficult to purify. We report a new method for obtaining pure D from urine of patients with Fanconi's syndrome. The method is simple and allows the purification of milligram amounts of D within a few days. It involves three chromatographic steps using Bio-Rex 70, hydroxylapatite HPLC, and reverse-phase HPLC. Protein D purified by this method is suitable for both functional and structural studies.

Chromatography, High Pressure Liquid↗
PubMed1984

Attachment and phagocytosis by salmon macrophages of agarose beads coated with human C3b and C3bi.

Agarose beads (diameter 5-10 micron) preincubated in human serum became associated (attached and ingested) to 50-60% of the salmon macrophages within 60 minutes. However, Beads preincubated in serum treated with heating (50 degrees C, 20 min) or with EDTA (10 mM) to inhibit the activation of alternative complement pathway, were not associated to the phagocytes. Furthermore, agarose beads coated with human C3b and C3bi after incubation with isolated complement factors (C3, D, B), were associated to 30-40% of the phagocytes. About 80% of the cell-associated agarose beads was intracellularly located. Conversion by trypsin treatment (0.01%) of agarose bound C3bi to C3d, abolished the association of such beads to the macrophages. The results demonstrate that salmon macrophages possess complement receptors that bind human C3b and C3bi. Agarose beads coated with these ligands (C3b and C3bi) are attached and ingested by the phagocytes.

Animals↗
PubMed1979

Limited proteolysis of complement components C2 and factor B. Structural analogy and limited sequence homology.

A method is described for the simultaneous purification of milligram quantities of complement components C2 and Factor B. Both products are homogeneous by the criteria of polyacrylamide-gel electrophoresis and N-terminal sequence analysis. Component C2 is cleaved by serine proteinase C1s at an X-Lys bond to give fragment C2a (approx. mol.wt. 74000) and fragment C2b (approx. mol.wt. 34000). The two fragments can be separated by gel filtration without the need for reducing or denaturing agents. Fragment C2b represents the N-terminal end of the molecule. Similar results were seen on cleavage of Factor B by Factor D in the presence of component C3. Again two non-covalently linked fragments are formed. The smaller, fragment Ba (approx. mol.wt. 36,000),) has threonine as the N-terminal residue, as does Factor B; the larger, fragment Bb (approx. mol. wt. 58000), has lysine as the N-terminal residue. A similar cleavage pattern is obtained on limited proteolysis of Factor B by trypsin, suggesting an Arg-Lys-or Lys-Lys bond at the point of cleavage. Although component C2 and Factor B show no apparent N-terminal sequence homology, a limited degree of sequence homology is seen around the sites of proteolytic cleavage.

Amino Acid Sequence↗
PubMed1994

The endogenous vascular elastase that governs development and progression of monocrotaline-induced pulmonary hypertension in rats is a novel enzyme related to the serine proteinase adipsin.

We showed previously a cause and effect relationship between increased activity of an endogenous vascular elastase (EVE) and experimentally induced pulmonary hypertension in rats. We now report the isolation and characterization of EVE. Degenerate oligonucleotides synthesized to homologous sequences in serine elastases were used in a PCR with rat pulmonary artery (PA) cDNA. The PCR product hybridized to a 1.2-kb mRNA and the intensity of hybridization was threefold increased in RNA from rat hypertensive PA at a timepoint when EVE activity was increased. The PCR product was used to screen a cDNA library and sequences obtained encoded rat adipsin. We then used immunoaffinity to purify EVE. An antibody to the elastin-binding protein was used to remove this competitor of elastase from the PA extract and the elastolytic activity increased 100-fold. The enzyme was purified using an antibody that recognizes NH2-terminal sequences of serine proteinases and the eluate was further purified using an antibody raised against recombinant adipsin. A single band at 20 kD immunoreactive with the adipsin antibody was resolved as an active enzyme on an elastin substrate gel. Immunogold labeling with an antibody to an adipsin peptide sequence localized EVE to PA smooth muscle cells. This is the first isolation of EVE; it appears to be a novel enzyme related to the serine proteinase adipsin originally found in adipose tissue.

Amino Acid Sequence↗