Genomic structure and promoter activity of the human tissue factor pathway inhibitor-2 gene.

Human type-2 tissue factor pathway inhibitor (TFPI-2), also known as placental protein 5, is a 32 kDa serine proteinase inhibitor consisting of three tandemly arranged Kunitz-type inhibitor domains homologous to tissue factor pathway inhibitor. TFPI-2 strongly inhibits a wide variety of serine proteinases including trypsin, chymotrypsin, plasmin, kallikrein and blood coagulation factor XIa. In this study, we have isolated and characterized a genomic clone from an artificial chromosome genomic library that encodes the entire human TFPI-2 gene. The human TFPI-2 gene spans approximately 7 kb and consists of five exons and four introns. Each Kunitz-type domain is encoded by a single exon, similar to that observed for murine TFPI-2 and other Kunitz-type proteinase inhibitors. A total of 535 bp of the 3'-flanking region contain two probable polyadenylation sites (AATAAA) at +4297 and +4314. A single transcription initiation site was identified by oligo-capping and reverse transcription-PCR analysis. Transient transfection of reporter plasmids containing segments of the 5'-flanking region into human transformed bone marrow endothelial cells and glioblastoma cells identified an 85 bp region (-224 to -139) sufficient for transcription of the human TFPI-2 gene.

Interleukin 21 and its receptor are involved in NK cell expansion and regulation of lymphocyte function.

Cytokines are important in the regulation of haematopoiesis and immune responses, and can influence lymphocyte development. Here we have identified a class I cytokine receptor that is selectively expressed in lymphoid tissues and is capable of signal transduction. The full-length receptor was expressed in BaF3 cells, which created a functional assay for ligand detection and cloning. Conditioned media from activated human CD3+ T cells supported proliferation of the assay cell line. We constructed a complementary DNA expression library from activated human CD3+ T cells, and identified a cytokine with a four-helix-bundle structure using functional cloning. This cytokine is most closely related to IL2 and IL15, and has been designated IL21 with the receptor designated IL21 R. In vitro assays suggest that IL21 has a role in the proliferation and maturation of natural killer (NK) cell populations from bone marrow, in the proliferation of mature B-cell populations co-stimulated with anti-CD40, and in the proliferation of T cells co-stimulated with anti-CD3.

Nucleotide sequence of the gene encoding murine tissue factor pathway inhibitor-2.

Tissue factor pathway inhibitor-2 (TFPI-2), also known as placental protein 5, is a 32 kDa extracellular matrix-associated serine proteinase inhibitor consisting of three tandemly-arranged Kunitz-type domains. Two overlapping genomic clones containing sequences encoding murine TFPI-2 were isolated from a lambda FIXII 129 SVJ mouse genomic library, and the complete nucleotide sequence of the gene was determined. The murine TFPI-2 gene spans approximately 9.3 kilobases and consists of five exons and four introns. The nucleotide sequences surrounding all the exon-intron boundaries are highly conserved and obey the GT-AG rule. Each Kunitz-type domain is encoded by a single exon, similar to that observed for other Kunitz-type proteinase inhibitors. A total of 1,577 bp of the 3'-flanking region contains a probable polyadenylation site (ATTAAA) at +5,759 and an apparent cleavage or termination site (CATTG) at +6,170. The 5'-flanking region of the murine TFPI-2 gene contains a prototypical TATA box, a GC box and two CAAT boxes. In addition, several candidate transcription factor binding sites responsible for placenta-, endothelial cell-, and smooth muscle cell-specific expression of the TFPI-2 gene were also identified.

Leptin does not fully account for the satiety activity of adipose tissue-conditioned medium.

To determine whether leptin alone accounts for the satiety activity secreted by native adipose tissue, we prepared culture media conditioned by microdissected adipose tissue from overfed Long-Evans rats, fa/fa rats, or db/db mice (media A, B, and C, respectively). Medium A significantly suppressed food intake following intracerebroventricular delivery to Long-Evans rats (2-h chow intake = 68 +/- 5% of baseline, P < 0.001). Media B and C significantly suppressed food intake following intraperitoneal delivery to ob/ob mice (24-h chow intake = 56 +/- 7% of baseline for medium B, P = 0. 001; 4-day chow intake = 78 +/- 3% of baseline for medium C, P = 0. 004). Using a leptin receptor-based bioassay, we determined that the leptin concentration of medium C was 392 +/- 18 ng/ml. This concentration was 20-fold lower than the concentration of recombinant murine leptin required to produce a similar degree of feeding suppression following 5 days of administration to ob/ob mice. Neither medium conditioned by adipose tissue from ob/ob mice nor medium conditioned by adipose tissue from fa/fa rats and subsequently immunodepleted of leptin had significant satiety activity. We conclude that leptin is necessary but not sufficient to account for the satiety activity of native adipose tissue, perhaps due to the production by adipocytes of a cofactor that augments the ability of leptin to suppress feeding.

Increased expression of mRNA for the long form of the leptin receptor in the hypothalamus is associated with leptin hypersensitivity and fasting.

The responsiveness of the hypothalamus to the inhibitory effects of leptin on food intake and body weight is influenced by multiple factors, including deficiency of either leptin or leptin receptors (Ob-R). To investigate whether altered expression of Ob-R in the hypothalamus could potentially contribute to altered leptin sensitivity, we performed in situ hybridization with riboprobes that detected either mRNAs encoding both the long (Ob-Rb) and short (Ob-Ra) splice variants or mRNA encoding only Ob-Rb. In the arcuate nucleus, mRNA encoding Ob-Rb, the predominant signaling form of the receptor, was 2.3 times greater in obese db/db and ob/ob mice than in lean +/ob controls (P < 0.01). In ob/ob mice, systemic administration of leptin reduced Ob-Rb mRNA content of the arcuate nucleus by 30% compared with saline-treated, pair-fed controls (P < 0.05). A 48-h fast increased Ob-Rb mRNA levels in the arcuate nucleus of normal and neuropeptide Y (NPY)-knockout mice (P < 0.01), although the effect was greater in the NPY-knockout mice (400 vs. 247%, P < 0.05). In addition, Ob-Rb mRNA hybridization was elevated by 40% in the arcuate nucleus (P < 0.05) and by 75% in the ventromedial nucleus (P < 0.05) of rats fasted 48 h. The results suggest that expression of Ob-Rb mRNA in the hypothalamus is sensitive to genetic and physiological interventions that alter circulating leptin levels, and that overexpression of Ob-Rb in the hypothalamus may contribute to increased leptin sensitivity.

Elevated free fatty acids induce uncoupling protein 3 expression in muscle: a potential explanation for the effect of fasting.

The newly described uncoupling protein 3 (UCP3) may make an important contribution to thermogenesis in humans because of its high level of expression in skeletal muscle. Contrary to expectations, fasting, a condition that reduces resting energy expenditure, has been reported to increase UCP3 expression in muscle. We have confirmed that a 10-fold increase in UCP3 mRNA levels occurs in rat quadriceps muscle between 12 and 24 h of food removal. A less consistent twofold increase in muscle UCP2 mRNA levels was observed in animals fasted for up to 72 h. Administration of recombinant leptin to prevent a fall in circulating leptin levels did not eliminate the fasting-induced increase in quadriceps UCP3 expression. Administration of a high dose of glucocorticoid to fed animals to mimic the increase in corticosterone induced by fasting did not reproduce the increase in UCP3 expression observed in fasted animals. In contrast, elevation of circulating free fatty acid levels in fed animals by Intralipid plus heparin infusion caused significant increases in the UCP3/actin mRNA ratio compared with saline-infused fed controls in both extensor digitorum longus (2.01 +/- 0.34 vs. 0.68 +/- 0.11, P = 0.002) and soleus muscles (0.31 +/- 0.07 vs. 0.09 +/- 0.02, P = 0.014). We conclude that free fatty acids are a potential mediator of the increase in muscle UCP3 expression that occurs during fasting. This seemingly paradoxical induction of UCP3 may be linked to the use of free fatty acid as a fuel rather than an increased need of the organism to dissipate energy.

Recombinant leptin exchanges between parabiosed mice but does not reach equilibrium.

Parabiosis experiments suggest that ob/ob mice are deficient in a circulating "lipostatic" signal but respond to such a signal when it is delivered in the cross circulation from their parabiotic partner. Identification of leptin as the mutation in ob/ob mice leads to the assumption that leptin is the lipostatic signal. The objective of these experiments was to determine the circulating half-life of leptin and to demonstrate whether it exchanged between parabiosed mice. Measurement of disappearance of recombinant leptin from serum in SWRJ mice indicated a circulating half-life of approximately 36 min. Single ob/ob mice or one member of a parabiosed pair of ob/ob mice received 50 micrograms recombinant murine leptin in two intraperitoneal injections a day for 10 days, starting 40 days after parabiosis surgery. Control mice and pairs received equivalent injections of vehicle. In single mice, leptin significantly reduced food intake, body weight, serum insulin, and pancreatic and liver weight. Leptin treatment of one member of a parabiosed pair of ob/ob mice reduced serum insulin, gut content (an index of food intake), and body fat in both partners. The injected parabiont lost more fat than its partner, and body temperature was increased only in the injected mouse, indicating that leptin did not reach equilibrium in the two animals. This was confirmed by Western blot analysis of serum leptin measured 2 h after injection. Therefore, although leptin can exchange between parabionts, its half-life is inadequate to allow equilibrium when a large concentration gradient exists between partners.

Effect of fasting, refeeding, and dietary fat restriction on plasma leptin levels.

The factors responsible for the variability in plasma leptin levels observed among individuals with similar body compositions remain unclear. To examine the impact of dietary variables, we compared the changes in leptin levels induced by fasting and dietary fat restriction with the expected decrease following a significant loss in adipose mass. A 21.4 +/- 3.7% weight loss led to a 76.3 +/- 8.1% decrease in mean plasma leptin level (25.2 +/- 9.3 to 6.1 +/- 3.4 ng/mL, P = 0.0001) in a group of 9 obese males. Despite a weight loss of only 2.6 +/- 0.8%, mean plasma leptin levels fell by 61.9 +/- 25.2% (8.5 +/- 4.5 to 2.4 +/- 0.5 ng/mL, P < 0.01) in 7 nonobese females subjected to 3 days of fasting. Leptin levels in fasted subjects returned to baseline within 12 h of refeeding. Individual high- and low-fat meals given to 19 subjects after an overnight fast had no effect on plasma leptin levels. Reduction in dietary fat content from 37-10% of total calories for 7 weeks was also without effect on plasma leptin levels in these subjects. We conclude that plasma leptin levels primarily reflect total adipose mass, rather than meal consumption or dietary energy source, but that the reduction in leptin levels with ongoing fasting is disproportionate to the reduction in adipose mass. The ability of fasting to deactivate this presumed physiological satiety system may have been advantageous in environments characterized by rapid changes in food availability.

Effect of regional fat distribution and Prader-Willi syndrome on plasma leptin levels.

Variability in the relationship of plasma leptin level to body mass index (BMI) could be caused by imperfect estimation of adipose mass by the BMI, heterogeneity in the pathogenesis of obesity in mixed subject groups, or variation in adipose tissue distribution. To investigate these possibilities, we examined the correlation of plasma leptin and BMI in an ethnically mixed population, a group of subjects with the Prader-Willi syndrome, and a group of Japanese-American subjects who underwent computerized tomographic measurement of adipose tissue cross-sectional areas. Highly significant and indistinguishable linear relationships between plasma leptin levels and BMI were found in the three study groups. Intersubject variability was also similar in the three groups and was reduced only when more accurate techniques for assessing adipose tissue mass were substituted for the BMI. The plasma leptin level of Japanese-American subjects in the highest quartile of intraabdominal fat area (mean area = 154.5 +/- 38.4 cm2) was 12.5 +/- 8.7 ng/mL as compared to 12.3 +/- 9.6 ng/mL (P = 0.91) for subjects in the lowest quartile of intraabdominal fat area (mean area = 51.2 +/- 20.1 cm2, P < 0.001 for difference in fat areas). We conclude that the circulating leptin level reflects total adipose tissue mass rather than a combination of adipose tissue mass and distribution, and that the Prader-Willi syndrome does not alter the relationship between these two variables.

Leptin is a metabolic gate for the onset of puberty in the female rat.

The timing of puberty onset in mammals is tightly coupled to the animal's nutritional and metabolic state. We conducted two experiments to test the hypothesis that leptin acts as a metabolic signal for the onset of puberty. In the first experiment, we administered leptin (6.3 micrograms/g twice daily) to a group of normal prepubertal female rats and compared their rate of sexual maturation to that of two control groups. The group of leptin-treated animals and one group of control animals were allowed to eat ad lib, while the other group of control animals was pair-fed to the leptin-treated group. Food intake in the leptin-treated group was reduced to approximately 80% of the ad lib-fed control group, resulting in retarded growth in both leptin-treated and pair-fed animals. All measured indices of pubertal maturation-age at vaginal opening, age at first estrus, ovarian weight, ovulatory index (corpora lutea/ovarian section), uterine weight, and uterine cross-sectional area-were significantly delayed in the pair-fed group but not different between the leptin-treated group and ad lib-fed controls. The second experiment was similar to the first, except that both the leptin-treated group and the pair-fed group were fed at 70% of the ad lib-fed controls. Under these conditions, leptin only partially reversed the delay in sexual maturation, as reflected by the age at vaginal opening and first estrus. These results suggest that leptin is not the primary signal that initiates the onset of puberty but that instead, it acts in a permissive fashion, as a metabolic gate, to allow pubertal maturation to proceed-if and when metabolic resources are deemed adequate; moreover, these observations suggest that other metabolic factors, besides leptin, influence the timing of puberty onset under conditions of more severe dietary stress.

Synergy between leptin and cholecystokinin (CCK) to control daily caloric intake.

Both cholecystokinin (CCK), a short-term meal-related satiety signal, and the ob protein leptin, a postulated long-term adiposity hormone, are thought to be important signals in the multiple interacting systems that control appetite and adiposity. We hypothesized that these hormones may synergistically interact to suppress feeding. Following IP administration of leptin (two doses of 50 micrograms each) and CCK (2, 4, 8, or 16 micrograms) total daily caloric intake was significantly reduced by leptin and CCK compared to leptin alone. These results support the hypothesis that CCK and leptin may synergistically interact to control long-term feeding.

Obesity genes and the regulation of body fat content.

Physiological investigation has demonstrated that the central nervous system monitors body composition and adjusts energy intake and expenditure to stabilize total adipose tissue mass. Genetic variations in the signalling molecules involved in this regulatory system account for the heritable component of body fat content. The application of molecular techniques to rodent models of Mendelian obesity has resulted in the characterization of five loci at which mutations produce an abnormal accumulation of body fat. The genes at these loci include agouti, which encodes a molecule that antagonizes the binding of alpha melanocyte-stimulating hormone to its receptor; fat, which encodes carboxypeptidase E; tubby, which encodes a putative phosphodiesterase; obese, which encodes a circulating satiety protein; and diabetes, which encodes the receptor for the obese gene product. A more detailed understanding of the functional interrelationships of these genes should lead to important new insights into the causes and potential therapies for human obesity.

Leptin is a metabolic signal to the reproductive system.

Leptin, a newly-discovered hormonal product of the obese (ob) gene, is expressed by adipocytes and thought to play a role in the regulation of food intake and metabolism. We tested the hypothesis that leptin signals metabolic information to the reproductive system by examining its effects on the reproductive system of ob/ob mice, which have a congenital deficiency in leptin and are infertile. We treated pair-fed males and females with leptin (50 microg twice daily, ip) or vehicle (n=10/group) for 14 days, after which the animals were bled and killed. Leptin-treated females had significantly elevated serum levels of LH, increased ovarian and uterine weights, and stimulated aspects of ovarian and uterine histology compared to controls. Leptin-treated males had significantly elevated serum levels of FSH, increased testicular and seminal vesicle weights, greater seminal vesicle epithelial cell height, and elevated sperm counts compared to controls. These results demonstrate that leptin stimulates the reproductive endocrine system in both sexes of ob/ob mice and suggest that leptin may serve as a permissive signal to the reproductive system of normal animals.

Specificity of leptin action on elevated blood glucose levels and hypothalamic neuropeptide Y gene expression in ob/ob mice.

Correction of the obese state induced by genetic leptin deficiency reduces elevated levels of both blood glucose and hypothalamic neuropeptide Y (NPY) mRNA in ob/ob mice. To determine whether these responses are due to a specific action of leptin or to the reversal of the obese state, we investigated the specificity of the effect of systemic leptin administration to ob/ob mice (n = 8) on levels of plasma glucose and insulin and on hypothalamic expression of NPY mRNA. Saline-treated controls were either fed ad libitum (n = 8) or pair-fed to the intake of the leptin-treated group (n = 8) to control for changes of food intake induced by leptin. The specificity of the effect of leptin was further assessed by 1) measuring NPY gene expression in db/db mice (n = 6) that are resistant to leptin, 2) measuring NPY gene expression in brain areas outside the hypothalamus, and 3) measuring the effect of leptin administration on hypothalamic expression of corticotropin-releasing hormone (CRH) mRNA. Five daily intraperitoneal injections of recombinant mouse leptin (150 micrograms) in ob/ob mice lowered food intake by 56% (P < 0.05), body weight by 4.1% (P < 0.05), and levels of NPY mRNA in the hypothalamic arcuate nucleus by 42.3% (P < 0.05) as compared with saline-treated controls. Pair-feeding of ob/ob mice to the intake of leptin-treated animals produced equivalent weight loss, but did not alter expression of NPY mRNA in the arcuate nucleus. Leptin administration was also without effect on food intake, body weight, or NPY mRNA levels in the arcuate nucleus of db/db mice. In ob/ob mice, leptin did not alter NPY mRNA levels in cerebral cortex or hippocampus or the expression of CRH mRNA in the hypothalamic paraventricular nucleus (PVN). Leptin administration to ob/ob mice also markedly reduced serum glucose (8.3 +/- 1.2 vs. 24.5 +/- 3.8 mmol/l; P < 0.01) and insulin levels (7,263 +/- 1,309 vs. 3,150 +/- 780 pmol/l), but was ineffective in db/db mice. Pair-fed mice experienced reductions of glucose and insulin levels that were < 60% of the reduction induced by leptin. The results suggest that in ob/ob mice, systemic administration of leptin inhibits NPY gene overexpression through a specific action in the arcuate nucleus and exerts a hypoglycemic action that is partly independent of its weight-reducing effects. Furthermore, both effects occur before reversal of the obesity syndrome. Defective leptin signaling due to either leptin deficiency (in ob/ob mice) or leptin resistance (in db/db mice) therefore leads directly to hyperglycemia and the overexpression of hypothalamic NPY that is implicated in the pathogenesis of the obesity syndrome.

Recombinant ob protein reduces feeding and body weight in the ob/ob mouse.

To determine whether the product of the recently cloned ob gene functions as an adipose-related satiety factor, recombinant murine ob protein was administered intraperitoneally to ob/ob mice. Monomeric ob protein given as single morning injections to groups of three animals at seven doses ranging from 5 to 100 micrograms reduced 24-h chow consumption in a dose-dependent manner from values of 81 +/- 6.8% of control (10-micrograms dose, P = 0.04) to 29 +/- 7.7% of control (100-micrograms dose, P < 0.0001). Daily injections of 80 micrograms of ob protein into six ob/ob mice for 2 wk led to an 11 +/- 1.6% decrease in body weight (P = 0.0009) and suppressed feeding to 26 +/- 4.9% of baseline (P < 0.0001), with significant reduction of serum insulin and glucose levels. The effect of recombinant ob protein on feeding was not augmented by cofactors secreted by adipose tissue, nor did exposure of adipose tissue to ob protein affect intracellular ob mRNA levels. Posttranslational modification of ob protein was not required for activity; however, addition of a hexahistidine tag to the amino terminus of the mature ob protein resulted in prolonged suppression of feeding after injection into ob/ob mice. These results demonstrate a direct effect of the ob protein to suppress feeding in the ob/ob mouse and suggest that this molecule plays a critical role in regulating total body fat content.

Human thrombopoietin: gene structure, cDNA sequence, expression, and chromosomal localization.

Thrombopoietin (TPO), a lineage-specific cytokine affecting the proliferation and maturation of megakaryocytes from committed progenitor cells, is believed to be the major physiological regulator of circulating platelet levels. Recently we have isolated a cDNA encoding a ligand for the murine c-mpl protooncogene and shown it to be TPO. By employing a murine cDNA probe, we have isolated a gene encoding human TPO from a human genomic library. The TPO locus spans over 6 kb and has a structure similar to that of the erythropoietin gene (EPO). Southern blot analysis of human genomic DNA reveals a hybridization pattern consistent with a single gene locus. The locus was mapped by in situ hybridization of metaphase chromosome preparations to chromosome 3q26-27, a site where a number of chromosomal abnormalities associated with thrombocythemia in cases of acute myeloid leukemia have been mapped. A human TPO cDNA was isolated by PCR from kidney mRNA. The cDNA encodes a protein with 80% identity to previously described murine TPO and is capable of initiating a proliferative signal to murine interleukin 3-dependent BaF3 cells expressing the murine or human TPO receptor.

Cloning and characterization of an abundant subtype of the human calcitonin receptor.

We have cloned and characterized a second form of the human calcitonin receptor from T47D cells. It resembles the clone described by Gorn et al. [J. Clin. Invest. 90:1726-1735 (1992)] except that it lacks a 16-amino acid insert in the putative first intracellular loop. The insert-negative receptor appears to be the most abundant form, and it occurs at a relatively constant level in all expressing tissues. In contrast, the insert-positive receptor is found at low levels in most tissues but its expression levels appear to be much more variable. The insert-negative cDNA was stably expressed in baby hamster kidney cells. Like the endogenous T47D receptor, the recombinant receptor has an equally high affinity for salmon and porcine calcitonin but a 3-4-fold lower affinity for human calcitonin. High concentrations of calcitonin gene-related peptide, rat amylin, secretin, or vasoactive intestinal peptide do not significantly compete with calcitonin for binding to the recombinant receptor. Calcitonin stimulates a cAMP response in both T47D and transfected baby hamster kidney cells. Salmon calcitonin is more potent than human calcitonin for T47D cells, but the two are nearly equipotent for the transfectants. Furthermore, the ED50 for the cAMP response in the transfectants is 10-100-fold lower than in T47D cells. Calcitonin stimulates inositol phosphate turnover and elevates internal calcium levels in the transfectants. This response requires non-physiological levels of calcitonin and is directly correlated with the number of receptors. Lastly, by using a human/rodent somatic cell hybrid panel and in situ hybridization, we localized the human calcitonin receptor gene to chromosome 7.

Cloning and expression of murine thrombopoietin cDNA and stimulation of platelet production in vivo.

The major regulator of circulating platelet levels is believed to be a cytokine termed thrombopoietin. It is thought to be a lineage-specific cytokine affecting the proliferation and maturation of committed cells resulting in the production of megakaryocytes and platelets. Despite considerable efforts by a number of laboratories, the unequivocal identification of thrombopoietin has proven elusive. Here we report the functional cloning of a murine complementary DNA encoding a ligand for the receptor encoded by the c-mpl proto-oncogene (c-Mpl). The encoded polypeptide has a predicted molecular mass of 35,000 (M(r) 35K). The protein has a novel two-domain structure with an amino-terminal domain homologous with erythropoietin and a carboxy-terminal domain rich in serine, threonine and proline residues and containing seven potential N-linked glycosylation sites. Intraperitoneal injections of mice with recombinant protein increase circulating platelet levels by greater than fourfold after 7 days. These results along with those presented in the accompanying report strongly suggest that the ligand for c-Mpl is thrombopoietin.

The human glucagon receptor encoding gene: structure, cDNA sequence and chromosomal localization.

Characterization of the human glucagon-receptor-encoding gene (GGR) should provide a greater understanding of blood glucose regulation and may reveal a genetic basis for the pathogenesis of diabetes. A cDNA encoding a complete functional human glucagon receptor (GGR) was isolated from a liver cDNA library by a combination of polymerase chain reaction and colony hybridization. The cDNA encodes a receptor protein with 80% identity to rat GGR that binds [125I]glucagon and transduces a signal leading to increases in the concentration of intracellular cyclic adenosine 3',5'-monophosphate. Southern blot analysis of human DNA reveals a hybridization pattern consistent with a single GGR locus. In situ hybridization to metaphase chromosome preparations maps the GGR locus to chromosome 17q25. Analysis of the genomic sequence shows that the coding region spans over 5.5 kb and is interrupted by 12 introns.