Absence of bacterially induced RELMbeta reduces injury in the dextran sodium sulfate model of colitis.

Although inflammatory bowel disease (IBD) is the result of a dysregulated immune response to commensal gut bacteria in genetically predisposed individuals, the mechanism(s) by which bacteria lead to the development of IBD are unknown. Interestingly, deletion of intestinal goblet cells protects against intestinal injury, suggesting that this epithelial cell lineage may produce molecules that exacerbate IBD. We previously reported that resistin-like molecule beta (RELMbeta; also known as FIZZ2) is an intestinal goblet cell-specific protein that is induced upon bacterial colonization whereupon it is expressed in the ileum and colon, regions of the gut most often involved in IBD. Herein, we show that disruption of this gene reduces the severity of colitis in the dextran sodium sulfate (DSS) model of murine colonic injury. Although RELMbeta does not alter colonic epithelial proliferation or barrier function, we show that recombinant protein activates macrophages to produce TNF-alpha both in vitro and in vivo. RELMbeta expression is also strongly induced in the terminal ileum of the SAMP1/Fc model of IBD. These results suggest a model whereby the loss of epithelial barrier function by DSS results in the activation of the innate mucosal response by RELMbeta located in the lumen, supporting the hypothesis that this protein is a link among goblet cells, commensal bacteria, and the pathogenesis of IBD.

Activation of SOCS-3 by resistin.

Resistin is an adipocyte hormone that modulates glucose homeostasis. Here we show that in 3T3-L1 adipocytes, resistin attenuates multiple effects of insulin, including insulin receptor (IR) phosphorylation, IR substrate 1 (IRS-1) phosphorylation, phosphatidylinositol-3-kinase (PI3K) activation, phosphatidylinositol triphosphate production, and activation of protein kinase B/Akt. Remarkably, resistin treatment markedly induces the gene expression of suppressor of cytokine signaling 3 (SOCS-3), a known inhibitor of insulin signaling. The 50% effective dose for resistin induction of SOCS-3 is approximately 20 ng/ml, close to levels of resistin in serum. Association of SOCS-3 protein with the IR is also increased by resistin. Inhibition of SOCS function prevented resistin from antagonizing insulin action in adipocytes. SOCS-3 induction is the first cellular effect of resistin that is independent of insulin and is a likely mediator of resistin's inhibitory effect on insulin signaling in adipocytes.

Adipose-derived resistin and gut-derived resistin-like molecule-beta selectively impair insulin action on glucose production.

The adipose-derived hormone resistin is postulated to link obesity to insulin resistance and diabetes. Here, the infusion of either resistin or the resistin-like molecule-beta (RELMbeta) rapidly induced severe hepatic but not peripheral insulin resistance. In the presence of physiologic hyperinsulinemia, the infusion of purified recombinant resistin, increasing circulating resistin levels by approximately twofold to 15-fold, inhibited glucose metabolism such that lower rates of glucose infusion were required to maintain the plasma glucose concentration at basal levels. The effects of resistin and RELMbeta on in vivo insulin action were completely accounted for by a marked increase in the rate of glucose production. These results support the notion that a novel family of fat- and gut-derived circulating proteins modulates hepatic insulin action.

Resistin promotes endothelial cell activation: further evidence of adipokine-endothelial interaction.

BACKGROUND: Adipocyte-derived hormones may represent a mechanism linking insulin resistance to cardiovascular disease. In the present study, we evaluated the direct effects of resistin, a novel adipocyte-derived hormone, on endothelial activation. METHODS AND RESULTS: Endothelial cells (ECs) were incubated with human recombinant resistin (10 to 100 ng/ML, 24 hours), and endothelin-1 (ET-1) release, ET-1 mRNA expression, and nitric oxide (NO) production were assessed. Transient transfection assays were used to evaluate the effects of resistin on transcription of human ET-1 gene promoter. Furthermore, the effects of resistin on AP-1-mutated ET-1 promoter were evaluated. The effects of resistin on expression of vascular cell adhesion molecule (VCAM-1) and monocyte chemoattractant chemokine (MCP-1) were studied in addition to CD40 receptor, CD40 ligand-induced MCP-1 expression, and tumor necrosis factor receptor-associated factor-3 (TRAF3), an inhibitor of CD40 signaling. Incubation of ECs with resistin resulted in an increase in ET-1 release and ET-1 mRNA expression, with no change in NO production. Whereas treatment with resistin resulted in an increase in ET-1 promoter activity, the AP-1-mutated promoter was inactive after resistin stimulation. Additionally, resistin-treated cells showed increased expression of VCAM-1 and MCP-1, with concomitant reductions in TRAF-3 expression. Resistin did not alter CD40 receptor expression; however, increased CD40 ligand induced MCP-1 production. CONCLUSIONS: The novel adipokine resistin exerts direct effects to promote EC activation by promoting ET-1 release, in part by inducing ET-1 promoter activity via the AP-1 site. Furthermore, resistin upregulates adhesion molecules and chemokines and downregulates TRAF-3, an inhibitor of CD40 ligand signaling. In this fashion, resistin may be mechanistically linked to cardiovascular disease in the metabolic syndrome.

Resistin promotes smooth muscle cell proliferation through activation of extracellular signal-regulated kinase 1/2 and phosphatidylinositol 3-kinase pathways.

BACKGROUND: Resistin, a novel adipokine, is elevated in patients with type 2 diabetes and may play a role in the vascular complications of this disorder. One recent study has shown that resistin has a proinflammatory effect on endothelial cells. However, there is no information on whether resistin could also affect vascular smooth muscle cells (SMCs). Thus, the purpose of this study was to assess whether resistin could induce SMC proliferation and to study the mechanisms whereby resistin signals in SMCs. METHODS AND RESULTS: Human aortic smooth muscle cells (HASMCs) were stimulated with increasing concentrations of resistin for 48 hours. Cell proliferation was induced by resistin in a dose-dependent manner as assessed by direct cell counting. To gain more insights into the mechanism of action of resistin, we investigated the extracellular signal-regulated kinase (ERK) and/or phosphatidylinositol 3-kinase (PI3K) signaling pathways. Transient phosphorylation of the p42/44 mitogen-activated protein kinase (ERK 1/2) occurred after addition of resistin to HASMCs. U0126, a specific inhibitor of ERK phosphorylation, significantly inhibited ERK 1/2 phosphorylation and reduced resistin-simulated proliferation of HASMCs. LY294002, a specific PI3K inhibitor, also significantly inhibited HASMC proliferation after resistin stimulation. CONCLUSIONS: Our results demonstrate that resistin induces HASMC proliferation through both ERK 1/2 and Akt signaling pathways. The proliferative action exerted by resistin on HASMCs may account in part for the increased incidence of restenosis in diabetes patients.

Adenovirus-mediated high expression of resistin causes dyslipidemia in mice.

The adipocyte-derived hormone resistin has been proposed as a possible link between obesity and insulin resistance in murine models. Many recent studies have reported physiological roles for resistin in glucose homeostasis, one of which is enhancement of glucose production from the liver by up-regulating gluconeogenic enzymes such as glucose-6-phosphatase and phosphoenolpyruvate carboxykinase. However, its in vivo roles in lipid metabolism still remain to be clarified. In this study, we investigated the effects of resistin overexpression on insulin action and lipid metabolism in C57BL/6 mice using an adenoviral gene transfer technique. Elevated plasma resistin levels in mice treated with the resistin adenovirus (AdmRes) were confirmed by Western blotting analysis and RIAs. Fasting plasma glucose levels did not differ between AdmRes-treated mice and controls, but the basal insulin concentration was significantly elevated in AdmRes-treated mice. In AdmRes-treated mice, the glucose-lowering effect of insulin was impaired, as evaluated by insulin tolerance tests. Furthermore, total cholesterol and triglyceride concentrations were significantly higher, whereas the high-density lipoprotein cholesterol level was significantly lower. Lipoprotein analysis revealed that low-density lipoprotein was markedly increased in AdmRes-treated mice, compared with controls. In addition, in vivo Triton WR-1339 studies showed evidence of enhanced very low-density lipoprotein production in AdmRes-treated mice. The expressions of genes involved in lipoprotein metabolism, such as low-density lipoprotein receptor and apolipoprotein AI in the liver, were decreased. These results suggest that resistin overexpression induces dyslipidemia in mice, which is commonly seen in the insulin-resistant state, partially through enhanced secretion of lipoproteins.

Leptin and adiponectin stimulate the release of proinflammatory cytokines and prostaglandins from human placenta and maternal adipose tissue via nuclear factor-kappaB, peroxisomal proliferator-activated receptor-gamma and extracellularly regulated kinase 1/2.

Beyond their effects on central metabolic functions, leptin, resistin, and adiponectin have profound effects on a number of other physiologic processes, including immune function and inflammation. Although leptin, resistin, and adiponectin are produced in human placenta and adipose tissue, their immunoregulatory actions in these tissues are not known. Therefore, the aim of this study was to determine the effect of leptin, resistin, and adiponectin on the release of proinflammatory mediators in human placenta and sc adipose tissue. Samples were obtained from normal pregnancies at the time of cesarean section. Tissue explants (n = 5) were incubated in the absence (basal control) or presence of a leptin (1, 10, and 100 ng/ml), resistin (1, 10, and 100 ng/ml), and adiponectin (0.1 and 0.5 microg/ml). After 6 h incubation, the medium was collected, and the release of IL-1beta, IL-6, TNFalpha, prostaglandin (PG)F2alpha and PGE2 was quantified by ELISA. There was no effect of resistin on proinflammatory cytokine or prostaglandin release; however, leptin at 100 ng/ml and adiponectin at 0.1 and/or 0.5 microg/ml significantly increased the release of IL-1beta, IL-6, TNFalpha, and PGE2 from human placenta and adipose tissue. Although both leptin and adiponectin significantly increased PGF2alpha release from human placenta, there was no effect of these hormones on PGF2alpha release from adipose tissue. Furthermore, this leptin- and adiponectin-induced proinflammatory response could be abrogated by treatment with the antiinflammatory ERK1/2 MAPK inhibitor U0126, the peroxisomal proliferator-activated receptor-gamma ligand troglitazone, and the nuclear factor-kappaB inhibitor BAY 11-7082. Collectively, these data indicate that leptin and adiponectin activate proinflammatory cytokine release and phospholipid metabolism in human placenta and adipose tissue, and antiinflammatory agents can abrogate leptin- and adiponectin-induced inflammation.

Recombinant human FIZZ3/resistin stimulates lipolysis in cultured human adipocytes, mouse adipose explants, and normal mice.

Human FIZZ3 (hFIZZ3) was identified as an ortholog of mouse resistin (mResistin), an adipocyte-specific secreted factor linked to insulin resistance in rodents. Unlike mResistin, hFIZZ3 is expressed in macrophages and monocytes, but is undetectable in adipose tissue. The profound macrophage infiltration of adipose that occurs during obesity suggests that hFIZZ3 may play an important role in adipocyte biology. Using a recombinant protein produced in Escherichia coli, we report here that chronic treatment of cultured human adipocytes with hFIZZ3 results in hypotropic cells with smaller lipid droplets. Recombinant hFIZZ3 facilitates preadipocyte proliferation and stimulates adipocyte triglyceride lipolysis, whereas recombinant mResistin inhibits adipocyte differentiation, with no detectable effect on proliferation or lipolysis. In addition, insulin-stimulated glucose uptake and Akt phosphorylation are not altered in hFIZZ3-treated adipocytes, indicating an intact insulin response. In mouse adipose explants, hFIZZ3 accelerates simultaneously triglyceride lipolysis and fatty acid reesterification, as assessed by measurement of glycerol and fatty acid release. Consistent with the in vitro findings, acute administration of recombinant hFIZZ3 into normal mice caused a significant increase in serum glycerol concentration with no elevation in free fatty acid at 45 min post injection. Taken together, the data suggest that recombinant hFIZZ3 can influence adipose metabolism by regulating preadipocyte cell number, adipocyte lipid content, and energy expenditure via accelerating the fatty acid/triglyceride futile cycle.

Resistin stimulation of 17alpha-hydroxylase activity in ovarian theca cells in vitro: relevance to polycystic ovary syndrome.

CONTEXT: A newly discovered hormone resistin has been shown to be increased in women with polycystic ovary syndrome (PCOS). OBJECTIVE: The purpose of this study was to confirm increased resistin concentrations in women with PCOS and to test the direct effect of resistin on human theca cell androgen production. DESIGN: Resistin was measured in fasting serum samples by RIA. To test the direct effects of resistin on ovarian androgen biosynthesis, human theca cells were cultured with resistin for 3 d in the presence and absence of forskolin and insulin. PATIENTS: Fasting serum samples were obtained from 45 women with PCOS and 74 regularly cycling premenopausal control women in the follicular phase of their menstrual cycles, and ovarian theca cell cultures were established from two control women. RESULTS: The mean serum resistin concentration was increased (40%) in women with PCOS. Serum resistin concentrations correlated positively with body mass index and testosterone in PCOS women but not in controls. There were no significant correlations between resistin and fasting insulin or indicators of insulin resistance when corrected for body mass index. In cultured human theca cells, basal 17alpha-hydroxylase activity was unchanged by resistin alone, but resistin enhanced 17alpha-hydroxylase activity in the presence of forskolin or a combination of forskolin plus insulin. Resistin (> or =1 ng/ml) augmented forskolin and forskolin plus insulin stimulation of CYP17 mRNA expression in a concentration-dependent manner. CONCLUSION: These data indicate that abnormal resistin secretion in PCOS may play a role in causing ovarian hyperandrogenism.

Central administration of resistin promotes short-term satiety in rats.

OBJECTIVE: Several hormones expressed in white adipose tissue influence food intake at the central level. We sought to determine whether resistin, a circulating adipose-derived hormone in rodents, has actions on the hypothalamus by determining the effects of central resistin injection on food intake and on hypothalamic Fos protein expression. DESIGN: As resistin expression in adipose tissue is influenced by altered nutritional status, we studied the effect of central resistin in both fed and pre-fasted rats. RESULTS: In fasted rats, central injection of resistin decreased food intake acutely and increased the number of cells that express Fos protein in the arcuate nucleus but not in any other hypothalamic structure. The effect on food intake was dose-dependent and did not result in the formation of a conditioned taste aversion. CONCLUSIONS: Taken together, these results provide the first evidence documenting a central action of resistin, which could be involved in a feedback loop targeting the hypothalamus. On the other hand, since we observed resistin mRNA in the arcuate and ventromedial nuclei of the hypothalamus, it is also possible that brain-derived resistin serves as a neuropeptide involved in the regulation of energy homeostasis. However, since resistin-induced satiety was modest and transient, as central administration for several days did not affect body weight, the physiological relevance and therapeutic potential of the observed principal phenomenon may be limited.

Effects of adipokines on expression of adrenomedullin and endothelin-1 in cultured vascular endothelial cells.

Obesity is a major risk factor for the development of hypertension. Adipokines may cause hypertension by acting both centrally and directly on the vascular vessels. We wished to clarify whether three adipokines, leptin, resistin and tumor necrosis factor-alpha, affect expression of adrenomedullin and endothelin-1 in vascular endothelial cells. Human umbilical vein endothelial cells were cultured for 24 h with leptin (1-10 nmol/l), resistin (1-10 nmol/l) or tumor necrosis factor-alpha (1-10 ng/ml). Expression of adrenomedullin and endothelin-1 was examined by radioimmunoassay and northern blot analysis. Immunoreactive-adrenomedullin in the medium and adrenomedullin mRNA expression levels were decreased by treatment of tumor necrosis factor-alpha time- and dose-dependently, whereas endothelin-1 secretion was not significantly changed by it. Leptin or resistin had no significant effects on expression of adrenomedullin or endothelin-1 in human umbilical vein endothelial cells. Under hypoxic conditions (1% O2), expression of both adrenomedullin and endothelin-1 was induced in these cells. Immunoreactive-adrenomedullin levels in the medium were decreased by treatment of tumor necrosis factor-alpha under hypoxia. Leptin or resistin had no significant effects on adrenomedullin or endothelin-1 expression also in hypoxia. These findings have raised the possibility that decreased expression of adrenomedullin by tumor necrosis factor-alpha may be related to the increased risk of hypertension and other cardiovascular diseases in obese subjects.

Resistin and type 2 diabetes: regulation of resistin expression by insulin and rosiglitazone and the effects of recombinant resistin on lipid and glucose metabolism in human differentiated adipocytes.

Resistin, an adipocyte secreted factor, has been suggested to link obesity with type 2 diabetes in rodent models, but its relevance to human diabetes remains uncertain. Although previous studies have suggested a role for this adipocytokine as a pathogenic factor, its functional effects, regulation by insulin, and alteration of serum resistin concentration by diabetes status remain to be elucidated. Therefore, the aims of this study were to analyze serum resistin concentrations in type 2 diabetic subjects; to determine the in vitro effects of insulin and rosiglitazone (RSG) on the regulation of resistin, and to examine the functional effects of recombinant human resistin on glucose and lipid metabolism in vitro. Serum concentrations of resistin were analyzed in 45 type 2 diabetic subjects and 34 nondiabetic subjects. Subcutaneous human adipocytes were incubated in vitro with insulin, RSG, and insulin in combination with RSG to examine effects on resistin secretion. Serum resistin was increased by approximately 20% in type 2 diabetic subjects compared with nondiabetic subjects (P = 0.004) correlating with C-reactive protein. No other parameters, including adiposity and fasting insulin levels, correlated with serum resistin in this cohort. However, in vitro, insulin stimulated resistin protein secretion in a concentration-dependent manner in adipocytes [control, 1215 +/- 87 pg/ml (mean +/- SEM); 1 nM insulin, 1414.0 +/- 89 pg/ml; 1 microM insulin, 1797 +/- 107 pg/ml (P < 0.001)]. RSG (10 nM) reduced the insulin-mediated rise in resistin protein secretion (1 nM insulin plus RSG, 971 +/- 35 pg/ml; insulin, 1 microM insulin plus RSG, 1019 +/- 28 pg/ml; P < 0.01 vs. insulin alone). Glucose uptake was reduced after treatment with 10 ng/ml recombinant resistin and higher concentrations (P < 0.05). Our in vitro studies demonstrated a small, but significant, reduction in glucose uptake with human recombinant resistin in differentiated preadipocytes. In human abdominal sc adipocytes, RSG blocks the insulin-mediated release of resistin secretion in vitro. In conclusion, elevated serum resistin in human diabetes reflects the subclinical inflammation prevalent in type 2 diabetes. Our in vitro studies suggest a modest effect of resistin in reducing glucose uptake, and suppression of resistin expression may contribute to the insulin-sensitizing and glucose-lowering actions of the thiazolidinediones.

Estrogen induced growth of human breast cancer cells (MCF-7) in athymic nude mice is enhanced by secretions from a transplantable pituitary tumor.

MCF-7, a human breast carcinoma cell line, was maintained s.c. in female athymic nude mice for a period of 5-6 weeks. Administration of estrogen (s.c. pellet of 17 beta-estradiol and estrone in drinking water, 0.5 mg/l) to these mice resulted in sustained (P less than 0.001) growth of MCF-7 tumors. Grafting of a prolactin and growth hormone secreting rat pituitary tumor to the estrogen-treated mice resulted in an increased (P less than 0.05) rate of MCF-7 tumor growth. MCF-7 did not grow in athymic nude mice grafted with rat pituitary tumor alone or in mice without hormone treatment (controls). Thus, secretions of pituitary hormones alone are not capable of promoting in vivo growth of MCF-7 although such secretions significantly enhance estrogen-induced growth of this cell line. A synergism between pituitary hormones and estrogen for in vivo growth of a human breast carcinoma has been demonstrated in this study.

Resistin promotes 3T3-L1 preadipocyte differentiation.

OBJECTIVE: To investigate the relationship between resistin (a potential link between obesity and type 2 diabetes) and preadipocyte differentiation. DESIGN: A rat resistin expression vector was transfected into 3T3-L1 preadipocytes and differentiation was compared between normal 3T3-L1 cells, rat resistin-transfected cells and non-transfected cells grown in conditioned medium taken from resistin-expressing cultures. METHODS: The rat resistin gene was inserted into the pDual GC and pEFGP-N2 expression vectors for examination of the effects of resistin overexpression in 3T3-L1 cells before and after differentiation was stimulated with 3-isobutyl-1-methyxanthine (MIX), insulin and dexamethasone (DEX). Smaller conserved fragments were inserted into short interference RNA (siRNA) expression vectors, for examination of the effect of targeted resistin inhibition on differentiation of resistin-overexpressing 3T3-L1 cells. RESULTS: Prior to stimulation, the resistin-transfected 3T3-L1 cells contained many more small lipid droplets than did non-transfected 3T3-L1 cells. Following stimulation, differentiation in the resistin-transfected 3T3-L1 cells was dramatically promoted, especially in the early stages. Stimulation of differentiation was also observed in non-transfected 3T3-L1 cells grown in resistin protein-containing conditioned medium. The expression of adipocyte differentiation-associated markers such as CCAAT enhancer binding protein (C/EBPalpha), retinoid X receptor (RXRalpha) and lipoprotein lipase (LPL) was upregulated in resistin-overexpressing cells, whereas expression of preadipocyte factor-1 (Pref-1), an inhibitor of preadipocyte differentiation, was downregulated. In addition, expression of two of the three tested siRNAs inhibited the adipoconversion process, providing further evidence that resistin promotes the differentiation of preadipocytes to adipocytes. CONCLUSION: Resistin can promote preadipocyte differentiation. Based on this, we propose that resistin may be an important candidate mediator of obesity-induced insulin resistance.

Differential expression of adrenomedullin and resistin in 3T3-L1 adipocytes treated with tumor necrosis factor-alpha.

DESIGN: It has recently been shown that deficiency of adrenomedullin (AM), a potent vasodilator peptide, leads to insulin resistance. We studied expression of AM in NIH 3T3-L1 adipocytes and compared it with expression of resistin, an adipocyte-derived peptide hormone that is proposed to cause insulin resistance. Moreover, we studied the effects of tumor necrosis factor-alpha (TNF-alpha), a known mediator of insulin resistance, on the expression of AM and resistin in 3T3-L1 adipocytes. METHODS: 3T3-L1 cells were induced to differentiate to adipocytes by insulin, dexamethasone and 3-isobutyl-1-methylxanthine. Expression of AM mRNA and resistin mRNA was examined by Northern blot analysis. Immunoreactive AM in the medium was measured by RIA. RESULTS: AM mRNA was expressed in preadipocytes, but barely detectable in adipocytes. Immunoreactive AM was detected in the medium of both preadipocytes and adipocytes, with about 2.5 times higher levels found in preadipocytes. In contrast, resistin mRNA was expressed in adipocytes, whereas it was not detected in preadipocytes. Treatment with TNF-alpha increased AM expression in both adipocytes and preadipocytes, whereas it decreased resistin mRNA levels in adipocytes. CONCLUSIONS: The present study has shown that AM expression was down-regulated and resistin expression was up-regulated during adipocyte differentiation of 3T3-L1 cells. TNF-alpha acted as a potent negative regulator of resistin expression and a potent positive regulator of AM expression in adipocytes, raising the possibility that in addition to its known actions in causing insulin resistance, TNF-alpha may also have actions against insulin resistance through AM and resistin.

Glucose metabolism during the menstrual cycle. Glucose tolerance during the one cycle and under the effect of ovulatory suppressants.

PIP: To investigate possible diabetogenic effects of endogenous and exogenous hormones, an intravenous glucose tolerance test was administered to 3 groups of women: those with a normal cycle (15), those on Sequalan sequential therapy (12), or those on progestational therapy (13). None had received oral contraceptives previously. The test was performed for every woman at the estrogen peak (day 13, 14, or 15) and at the progesterone peak (day 23, 24, or 25) of a 28-day cycle. For each of the three groups there was no significant glucose metabolism difference between the two phases of the cycle. Estrogens could still reduce glucose tolerance if the luteal peak of urinary estrogen secretion, which occurs in most cycles, is characterized by the presence of nonbiologic estrogen precursors for estriol. Glucose tolerance can be investigated at any part of the cycle.^ieng

Resistin, but not adiponectin, inhibits dopamine and norepinephrine release in the hypothalamus.

Adiponectin (Adipocyte Complement-Related Protein of 30 kDa, ACRP30) and resistin are adipocyte-derived polypeptide hormones playing a role in metabolic homeostasis. Their plasma levels are inversely (adiponectin) or directly (resistin) correlated to obesity and they have opposite effects on insulin sensitivity. Adipose tissue hormones such as leptin have been shown to modulate neurotransmitters which control feeding in the hypothalamus. We have studied the effects of adiponectin and resistin on dopamine, norepinephrine and serotonin release from hypothalamic neuronal endings (synaptosomes) in vitro. We have found that adiponectin does not modify either basal or depolarization-induced amine release, while resistin inhibits the stimulated release of dopamine and norepinephrine, leaving unaffected serotonin release. We can conclude that, similarly to leptin, but differently from adiponectin, the adipose tissue hormone resistin could affect the central mechanisms of feeding by inhibiting catecholamine release in the hypothalamus.

Production and characterization of bioactive recombinant resistin in Escherichia coli.

Type 2 diabetes, characterized by peripheral target tissue resistance to insulin, is epidemic in industrialized countries and is strongly associated with obesity. The protein hormone, resistin, secreted specifically by the adipose tissues, is found to antagonize insulin action upon glucose uptake and may serve as an important role between human obesity and insulin resistance. Here, we report the production of bioactive recombinant resistin in Escherichia coli. cDNA of resistin was obtained by RT-PCR from mRNA of mouse differentiated NIH/3T3-L1 cells. The cDNA of mature resistin was inserted in the pQE-31 vector and the recombinant plasmid was transferred into E. coli JM109. After IPTG induction, the rec. resistin found in the inclusion body was dissolved in 6 M guanidine-HCl in the presence of 10 mM beta-mercaptoethanol. The His-tag containing protein was purified by Ni-NTA column to 95% homogeneity. After a quasi-static-like refolding process, the secondary structure of the rec. resistin was elucidated by circular dichroism which indicated that the protein was composed of 34.3% alpha-helix, 8.9% beta-sheet, 23.4% beta-turn, and 31.2% unordered structure. No disulfide-linked homodimers were formed in SDS-PAGE analysis under non-reducing conditions. The rec. resistin showed a dose-dependent antagonizing action against insulin in [3H]-2-deoxy-glucose transport in a broad range from 1 ng ml(-1) to 10 microg ml(-1) of resistin. A suppression of 85% of transport was achieved at the dosage of 10 microg ml(-1). This result may indicate that the rec. resistin does not need to form homodimers to establish its bioactivity. The rec. resistin will be useful for exploring the biological functions of this newly discovered hormone.

The pathophysiology of secretory diarrheas.

The mechanisms by which bacterial enterotoxins cause secretory diarrheas have been well defined, and the definitions of such mechanisms have been important in developing a consistently successful therapeutic approach. The less common secretory diarrheas, caused by the interaction of hormones of tumor origin with the gut small intestinal mucosa have also been clearly defined, and their pathogenetic mechanisms are similar to those by which the cholera and E. coli enterotoxins cause secretory diarrhea. The mechanisms by which histamine and gastrin of tumor origin cause gastric hypersecretion are less clearly delineated; secretory diarrhea caused by both of these agents can be stopped by total gastrectomy without removal of the responsible tumor. The secretory diarrhea caused by villous adenomas of the colon, which does not appear to be related to a distally produced humoral agent, results in the same picture of hypokalemic acidosis that is characteristic of the nonbacterial secretory diarrheas originating in the small intestine and is cured by resection of the responsible tumor.

Paracrine effect of human chorionic gonadotropin ectopically produced from papillary thyroid cancer cells on growth and function of FRTL-5 rat thyroid cells.

It is well known that human chorionic gonadotropin (hCG) is sometimes secreted from nontrophoblastic neoplasms. To elucidate the role of ectopic hCG, we investigated the effect of hCG produced from a papillary thyroid cancer cell line (B-CPAP cells) on stimulation and growth promotion of FRTL-5 rat thyroid cells. Ectopic hCG contained in the culture medium of B-CPAP cells was purified using gel filtration and bioassayed for thyrotropic activity in FRTL-5 cells. Addition of ectopic hCG (up to 5.2 x 10(4) IU/L) increased cyclic adenosine monophosphate (cAMP) accumulation and 3H-thymidine incorporation in FRTL-5 cells dose dependently. These effects were almost as potent as the stimulation induced by standard hCG CR-127. After the absorption of the ectopic hCG by anti-hCG-beta monoclonal antibody, the cAMP accumulation was significantly decreased. Analysis of ectopic hCG isoforms with different isoelectric points indicated the predominance of the acidic hCG isoform with isoelectric point (pI) 3.8-3.2 that is the major isoform of standard hCG. Basic isoforms (pI 5.7-5.3) with higher thyrotropic potency were also detected. These results indicate that the ectopic hCG secreted from papillary thyroid cancer cells possess intrinsic thyroid-stimulating and growth-promoting activity. The ectopic hCG may act as an autocrine-paracrine factor in nontrophoblastic neoplasms.