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Adipsin, a serine protease homolog, is synthesized and secreted by adipose cells and is found in the bloodstream. The expression of adipsin messenger RNA (mRNA) and protein was analyzed in rodents during metabolic perturbations and in several experimental models of obesity. Adipsin mRNA abundance is increased in adipose tissue during fasting in normal rats and in diabetes due to streptozotocin-induced insulin deficiency. Adipsin mRNA abundance decreased during the continuous infusion of glucose, which induces a hyperglycemic, hyperinsulinemic state that is accompanied by an increased adipose mass; it is suppressed (greater than 100-fold) in two strains of genetically obese mice (db/db and ob/ob), compared to their congenic counterparts, and is also reduced when obesity is induced chemically by injection of monosodium glutamate into newborn mice. Circulating adipsin protein is decreased in these animal models of obesity, as determined by immunoblotting with antisera to adipsin. Little change in adipsin expression is observed in a model of obesity obtained by pure overfeeding of normal rats (cafeteria model). These data suggest a possible role for adipsin in the above-mentioned disordered metabolic states, and raise the possibility that adipsin expression may be used to distinguish obesities that arise from certain genetic or metabolic defects from those that result from pure overfeeding.

Adipsin expression at the protein and mRNA levels is greatly reduced in several distinct syndromes of obesity in the mouse: genetic obesity due to the db/db and ob/ob genes, and a chemically induced model secondary to neonatal exposure to monosodium glutamate. We considered first the possibility that the adipsin gene might be identical to the db or ob locus and the lowered expression of this protein might result from a mutation in this gene. We show here that the adipsin structural gene is located on chromosome 10 and hence is physically distinct from any obesity genes so far identified in the mouse. A major role for the adrenal gland and adrenal glucocorticoids in the aberrant regulation of adipsin in these models of obesity is indicated by several experiments. Adrenalectomy of the ob/ob mouse raises the circulating levels of adipsin protein and the amount of this mRNA in epididymal fat pads (5-fold), although neither is increased to the levels seen in lean controls. Exogenous administration of corticosterone completely blocks the effects of adrenalectomy on adipsin, suggesting that the effect of this endocrine ablation is through reduction of adrenal glucocorticoids. Corticosterone administration also causes suppression in the levels of adipsin mRNA and protein in lean mice, although this decrease is never as severe as that seen in obese mice. The effect of exogenous corticosterone in lean mice occurs within 2 days and hence is not secondary to the obesity which these hormones eventually elicit. These results indicate that glucocorticoids can regulate adipsin expression in vivo and strongly suggest that the hyperglucocorticoid state seen in certain obese models plays a significant role in lowering adipsin mRNA and protein levels. Quantitative analysis of these experiments suggests that other as yet unknown neuroendocrine factors also function to suppress adipsin in obesity.

Adipsin gene expression as assessed by mRNA amounts was examined in adipose tissue of genetically obese rats at the onset (16 days of age) or at later stages (30 and 60 days of age) of obesity. Amounts of mRNA were equivalent in obese and lean rats at 16 days of age. In adult rats, we observed a 2-fold decrease in adipsin mRNA in the obese rats compared with control lean rats, which was abolished by weaning the animals on a high-fat diet. Our data show that, in sharp contrast with genetically obese mice, adipsin mRNA is not suppressed in genetically obese Zucker rats.

We examined the effects of RU38486, a potent glucocorticoid and progestin antagonist, upon several aspects of 3T3-F442A adipocyte differentiation. RU38486 accelerated the onset of differentiation, as monitored by cell morphological changes, accumulation of lipid droplets and widespread increases in the rate of expression of several enzyme adipose markers and specific mRNAs. RU38486, at a maximal concentration of 1 microM, dramatically hastened the emergence of both fatty-acid synthetase (FAS) and glycerol-3-phosphate dehydrogenase (G3PDH) enzyme activities (550% and 450% above control values 4 days after confluence, respectively). RU38486 induction of G3PDH-specific activity ran parallel to an increase in G3PDH mRNA content (2.4-fold the control content 4 days after confluence). Moreover, RU38486-treated cells exhibited enhancement of adenylate cyclase sensitivity to both isoproterenol and ACTH (160% and 350% above control activities 8 days after confluence, respectively). While the level of expression of lipogenic markers reached similar values at the mature stage, RU38486 enabled cells to acquire hypersensitivity in terms of ACTH-stimulated adenylate cyclase activity. Similarly, adipsin gene expression was highly potentiated by the drug at day 15 post-confluence (5-fold the control value). RU38486 responsiveness observed in differentiating 3T3-F442A cells is dependent upon their prior developmental activation; none of the studied markers could be induced by the drug in the undifferentiating 3T3-C2 cell subclone. Finally, this antiglucocorticoid appears to be a useful tool for studies on adipose conversion in vitro; it could permit a re-evaluation of the role of glucocorticoids in the understanding of adipocyte development.

Adipocytes constitute a major part of the bone marrow stroma in vivo and may play an active role in lymphohematopoiesis. Earlier studies had shown that the bone marrow stromal cell clone BMS2 was capable of adipocyte differentiation in vitro, in addition to its well-defined ability to support B lymphopoiesis. We now demonstrate that the process of adipogenesis in this functional bone marrow stromal cell clone can be inhibited by the cytokines interleukin-1 alpha, tumor necrosis factor, and transforming growth factor beta. Exposure of preadipocyte BMS2 cells to these agents blocked the induction of adipocyte differentiation as assessed by morphologic criteria and analysis of the neutral lipid content. Both interleukin-1 alpha and tumor necrosis factor elicited a rapid transient elevation in the steady-state mRNA levels of c-fos, c-jun, and JE. When added to differentiated adipocytes, the three cytokines continued to act as adipogenic antagonists. This was indicated by concentration- and time-dependent decreases in the activity of an adipocyte-specific enzyme, lipoprotein lipase. These changes in enzyme activity correlated directly with a decrease in steady-state levels of lipoprotein lipase mRNA. Another RNA marker of adipocyte differentiation (adipsin) was less influenced by the adipogenic antagonists. This may reflect the longer half-life of this mRNA transcript compared with those of lipoprotein lipase. Our results dramatically demonstrate that the differentiation state of bone marrow stromal cells can be modulated by exogenous factors in vitro. It is also the first report that transformation growth factor beta regulates the activity of lipoprotein lipase. These data suggest potential physiologic actions for these cytokines in vivo within the overall context of lymphohematopoiesis.

Highly purified human factor B of the alternative complement pathway was treated with factor D in the presence of cobra venom factor to generate its Ba and Bb cleavage fragments. These cleavage fragments were isolated by preparative polyacrylamide gradient gel electrophoresis followed by electrodialysis elution and treatment with CNBr. The resultant CNBr cleavage peptides were isolated by molecular exclusion high performance liquid chromatography and characterized by SDS polyacrylamide gel electrophoresis. Results of these experiments indicate that the Ba fragment essentially consisted of a 28,000 CNBr peptide, whereas 34,700 (28,000 + 3,500 when characterized under reducing polyacrylamide gel electrophoresis conditions); 14,500 (=20,000); and 8,300 CNBr peptides were derived from the Bb fragment.

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.

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.

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.

The carbohydrate composition of the second, third and fifth components of human complement (C2, C3 and C5) and of factors B and D was determined employing gas-chromatographic and mass-spectrometric methods. C2 was found to contain 15.9% carbohydrate composed of fucose, galactose, mannose, N-acetylglucosamine and N-acetylneuraminate (approximate molar ratio 1:4:9:9:4). N-acetylglucosamine and mannose (approximate molar ratio 1:4), amounting to 1.7% of the mass of the molecule, were the only monosaccharides detected in C3. C5 contained 3.8% carbohydrate composed of galactose, mannose, N-acetylglucosamine and N-acetylneuraminate (approximate molar ratio 2:4:4-5:2). The carbohydrate moiety of B consisted of fucose, galactose, mannose, N-acetylglucosamine and N-acetylneuraminate (molar ratio 1:2:3:4:2). The total carbohydrate content of B was estimated at 8.6%. In addition to these monosaccharides, glucose (0.4-0.9%) was also detected in all preparations analysed. Glucose was the only sugar detected in D.

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.

This article summarized the physicohemical data on the factors which compose the properdin system in guinea pig and man. The following other topics are discussed: (1) Activation of the properdin system; (2) Formation of the initiating and amplification C3 convertases; (3) Formation of the C5 convertase, and (4) Regulatory control mechanisms of the properdin system.

The C3 convertase of the C3b feedback cycle was generated in the fluid phase from C3b and factors B and D. It was shown to be an effective decomplementing agent when reacted with human serum. Many of the effects observed were dependent on the operation of the feedback during the ongoing reaction. Oxidization of factor B with iodine did not significantly increase the potency or the stability of the enzyme. Treatment of C3b with antrypol inhibited the formation of the enzyme. In preliminary experiments in the rat, the convertase was successfully formed in vivo, accompanied by cleavage of C3, consumption of C5, C6 and C7, and a biphasic change in the circulating neutrophils.

A cDNA library of Ob1771 preadipocytes was constructed, and a cDNA clone designated pOb24 was isolated by differential screening. The pOb24 mRNA, 6 kilobases in length, rose sharply in early differentiating Ob1771 and 3T3-F442A cells and decreased thereafter. In mouse adipose tissue, it was present at a high level in stromal-vascular cells (containing adipose precursor cells) and at a low level in mature adipocytes. Thus, pOb24 mRNA appears to be both in vitro and in vivo an unique marker of the preadipose state, i.e. of cell commitment during adipose cell differentiation. In contrast to glycerol-3-phosphate dehydrogenase mRNA, the emergence of pOb24 mRNA in Ob1771 cells required neither growth hormone or triiodothyronine as obligatory hormones nor insulin as a modulating hormone. Comparative studies of the expression of pOb24 and dihydrofolate reductase genes during the cell cycle suggest that arrest at the G1/S boundary was critical for the entry into the preadipose state. Tumor necrosis factor and transforming growth factor-beta were able to induce a large decrease of pOb24 mRNA level in growth-arrested Ob1771 cells. This decrease was shown to be only confined to early differentiating, glycerol-3-phosphate dehydrogenase negative cells as no decrease of pOb24 mRNA level was observed in glycerol-3-phosphate dehydrogenase positive cells. This result suggests that signals generated by tumor necrosis factor and transforming growth factor-beta have no effect on a commitment-related gene in late differentiated cells.

The mouse adipsin gene encodes a member of the serine protease family that is expressed predominantly in adipose tissue and is secreted into the bloodstream. Adipsin expression is sharply down-regulated in several models of genetic and acquired obesity, representing the first example of an adipocyte gene whose expression is greatly altered in this disorder. In this study, we have asked whether a DNA fragment from the adipsin gene can direct tissue-specific expression of a heterologous gene and mediate the suppression of this expression in genetic and chemically induced obesity. Transgenic mice have been constructed with 950 bases of DNA from the 5' flanking region of the adipsin gene linked to the bacterial chloramphenicol acetyltransferase (CAT) gene in a mouse strain bearing a recessive obesity gene (diabetes, db). By crossing db/+ transgenic mice with nontransgenic db/+ mice, we obtained progeny that allowed a direct comparison of CAT expression in the tissues of lean and obese littermates. The lean mice express CAT activity predominantly in adipose tissue, while the obese mice show a marked reduction in CAT expression relative to the lean controls. When similar experiments are performed with an adipsin-CAT fusion gene containing a heterologous AKV (AKR mouse leukemia virus) enhancer, the tissue specificity of CAT expression in lean mice is broadened to include the thymus, spleen, brain, and other tissues; down-regulation occurs in all of these tissues in mice homozygous for the obesity gene or in mice that have been injected with monosodium glutamate (MSG), which induces obesity. These results indicate that 950 bases of the 5' flanking region of the adipsin gene carry information that specifies both expression in adipose tissue and a response to a gene or chemical that induces obesity. These results also suggest that the trans-acting factors that are regulated aberrantly in these forms of obesity are not restricted to adipose tissue and could play a role in obesity-linked dysfunctions observed in other tissues as well.

The factors influencing the ultracentrifugation characteristics of immune complexes generated in the presence of fresh normal human serum have been analysed. In the absence of alternative pathway factors B, D or Properdin, the size of complexes was increased. When classical pathway function was blocked, in C1q deficient serum or in the presence of Mg EGTA, although the proportion of complexes remaining in solution were reduced their size was similar to those formed in normal human serum. In C2 deficient serum, a heterogeneous population of complexes was generated. In all instances repletion with the appropriate missing complement component reversed the abnormality. We conclude that there is normally a rapid sequential process of classical followed by alternative pathway activation leading to stable soluble complexes. In the absence of C1 activation the alternative pathway process requires precipitation of the antigen-antibody aggregates whereas in normal serum these events occur in the fluid phase. We suggest that in C2 deficient serum the C1 and/or C4 reacted complexes fail to activate the alternative pathway efficiently.

The release of adipsin, a serine proteinase with complement factor D activity, from 3T3-L1 adipocytes was measured by quantitative immunoblotting. This protein is secreted constitutively from 3T3-L1 adipocytes, and there is a 2-fold increase in the amount of adipsin released from cells treated with insulin for 1 to 10 min. Longer exposure to insulin had no further effect on the rate of adipsin release. Adipsin does not appear to be anchored by a glycosylphosphatidylinositol moiety, since adipsin which was been released with Triton X-114 from an intracellular membrane fraction partitions into the aqueous phase. Using a previously described procedure for the isolation of vesicles containing the insulin-responsive intracellular glucose transporters (GT vesicles), we show here that these GT vesicles contain an insulin-responsive pool of adipsin. Thus, insulin stimulates the secretion of a soluble protein, adipsin, as well as translocation to the plasma membrane of integral membrane proteins, including the glucose transporter, the transferrin receptors, and the insulin-like growth factor II receptor.

We have isolated, mapped and sequenced adipsin, the adipocyte differentiation-dependent serine protease gene. This gene, which is present in a single form in the mouse, spans 1.7 kilobases and contains five exons. While the basic exon structure characteristic of serine protease genes is conserved in adipsin, there is also a fusion of two exons that are separate in other serine proteases. The sequence data also suggests a mechanism of alternative splicing which appears to account for the generation of two adipsin mRNA species differing by only three nucleotides and encoding two different signal peptides. To investigate the control of adipsin expression we have examined the effects of tumor necrosis factor (TNF) on adipocytes. The level of adipsin RNA is dramatically decreased by hormone treatment, but the change occurs more slowly than for other fat cell mRNAs, such as glycerophosphate dehydrogenase. These results show that adipsin is a novel serine protease gene whose expression is regulated by a macrophage-derived factor which modulates expression of other adipocyte-specific RNAs.

Insulin is known to play the role of a positive effector both in vitro on the adipose conversion process and in vivo on the fatty acid synthesis and esterification processes in adipose tissue. The effects of insulin on the expression of two genes activated during adipose conversion, glycerol-3-phosphate dehydrogenase (GPDH) and adipsin genes, have been investigated in 3T3 F442A adipose cells. Within a physiological range of concentrations, insulin exerts opposite effects on the levels of GPDH (EC50 approximately 0.2 nM) and adipsin (EC50 approximately 1 nM) mRNAs. Its negative effect on the abundance of adipsin mRNA involves primarily a rapid inhibition of the transcriptional rate (less than 2 h). Its positive effect on the abundance of GPDH mRNA is due to a stimulation of the transcriptional rate accompanied by a delayed stabilization of GPDH mRNA. In addition, insulin exerts a specific effect on the length of the poly(A) tract of the adipsin mRNA. These results show that a single mechanism for the regulation of adipose-related genes by insulin can be excluded but rather suggest a complex phenomenon in which various levels of regulation take place.

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.

Factor D was isolated from human plasma by chromatography on CM-Sephadex C50, Sephadex G-75, and hydroxylapatite. Digestion of reduced, S-carboxymethylated factor D with cyanogen bromide resulted in three peptides which were isolated by chromatography on Sephadex G-75 (superfine) equilibrated in 20% formic acid. NH2-Terminal sequences were determined by automated Edman degradation with a Beckman 890C sequencer using a 0.1 M Quadrol program. The smallest peptide (CNBr III) consisted of the NH2-terminal 14 amino acids. The other two peptides had molecular weights of 17,000 (CNBr I) and 7000 (CNBr II). Overlap of the NH2-terminal sequence of factor D with the NH2-terminal sequence of CNBr I established the order of the peptides. The NH2-terminal 53 residues of factor D are somewhat more homologous with the group-specific protease of rat intestine than with other serine proteases. The NH2-terminal sequence of CNBr II revealed the active site serine of factor D. The typical serine protease active site sequence (Gly-Asp-Ser-Gly-Gly-Pro was found at residues 12-17. The region surrounding the active site serine does not appear to be more highly homologous with any one of the other serine proteases. The structural data obtained point out the similarities between factor D and the other proteases. However, complete definition of the degree of relationship between factor D and other proteases will require determination of the remainder of the primary structure.

The efficiency of some anti-tumour polysaccharides, such as lentinan, pachyman, pachymaran, carboxymethylpachymaran and hydroxyethylpachyman to trigger the alternative pathway of complement activation (APC) was investigated in detail, in order to clarify whether solid phase activation of APC by these polysaccharides will occur or not. From the eight polysaccharides tested, all were found to be potent activators of the alternative pathway except for carboxymethylpachymaran, regardless of their potency of inhibition of sarcoma 180 (S 180) transplanted in mice. This activation was observed both for the insoluble as well as the soluble part of the same polysaccharide. The turnover of C3, C5 and factor B showed no difference among these seven active polysaccharides. The polysaccharide particles (PX), isolated after treatment with C4d GPS showed prominent C3 consuming activity, which disappeared during prolonged incubation at 37 degrees. The activity of the decayed enzyme could be regenerated by treatment with the purified factor B. The stability of the particulate enzyme PX was comparable to the zymosan-complex (ZX) previously described.

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.