Serum resistin levels of obese and lean children and adolescents: biochemical analysis and clinical relevance.

OBJECTIVE: It was hypothesized that resistin links obesity with diabetes, but this has not been studied in children and adolescents to date. PATIENTS: We determined serum resistin levels of 135 obese (body mass index, 32.0 +/- 6.2 kg/m2; age, 12.6 +/- 3.4 yr) and 201 lean children (body mass index, 18.7 +/- 2.4 kg/m2; age, 12.5 +/- 2.5 yr) by a newly developed and extensively evaluated in-house immunoassay. These results were controlled for their association with markers of puberty, obesity, and insulin sensitivity. RESULTS: The analytical evaluation of our assay revealed different resistin isoforms with major peaks of higher than 660 and 55 kDa in the size exclusion chromatography. Using this assay system we found no difference in the resistin levels of obese compared with lean subjects (P = 0.48). However, resistin was significantly higher in girls than in boys (6.74 +/- 2.42 vs. 5.79 +/- 2.45; P < 0.001). Interestingly, in both obese and lean children, resistin correlated with age (P < 0.01), Tanner stage, and testosterone and estradiol levels (P < 0.05). In contrast, no significant correlation was found with parameters of insulin resistance such as homeostasis model assessment, insulin sensitivity index, or insulin, proinsulin, and glucose concentrations in obese subjects. CONCLUSIONS: Resistin appears to be not the main link between obesity and insulin resistance in children and adolescents but because of its association with Tanner stage, it may be related to the maturation of children during pubertal development. Additionally, we have demonstrated the presence of different molecular isoforms of resistin in human blood, and this may raise problems in comparing data from diverse assay systems.

An update on the biology and physiology of resistin.

Resistin is a newly discovered adipocyte hormone. It is related to resistin-like molecules alpha, beta and gamma in structure and function. Resistin is produced by white and brown adipose tissues but has also has been identified in several other tissues, including the hypothalamus, pituitary and adrenal glands, pancreas, gastrointestinal tract, myocytes, spleen, white blood cells and plasma. The tissue level of resistin is decreased by insulin, cytokines such as tumour necrosis factor alpha, endothelin-1 and increased by growth and gonadal hormones, hyperglycaemia, male gender and some proinflammatory cytokines, such as interleukin-6 and lipopolysaccharide. Resistin antagonizes insulin action, and it is downregulated by rosiglitazone and peroxisome proliferator-activated receptor gamma agonists. Since evidence of a direct link between resistin genotype and human diabetes is still weak, more molecular, physiological and clinical studies are needed to determine the role of resistin in the aetiology of type 2 diabetes.

Endocrine regulation of energy metabolism: review of pathobiochemical and clinical chemical aspects of leptin, ghrelin, adiponectin, and resistin.

BACKGROUND: Recent studies point to the adipose tissue as a highly active endocrine organ secreting a range of hormones. Leptin, ghrelin, adiponectin, and resistin are considered to take part in the regulation of energy metabolism. APPROACH: This review summarizes recent knowledge on leptin and its receptor and on ghrelin, adiponectin, and resistin, and emphasizes their roles in pathobiochemistry and clinical chemistry. CONTENT: Leptin, adiponectin, and resistin are produced by the adipose tissue. The protein leptin, a satiety hormone, regulates appetite and energy balance of the body. Adiponectin could suppress the development of atherosclerosis and liver fibrosis and might play a role as an antiinflammatory hormone. Increased resistin concentrations might cause insulin resistance and thus could link obesity with type II diabetes. Ghrelin is produced in the stomach. In addition to its role in long-term regulation of energy metabolism, it is involved in the short-term regulation of feeding. These hormones have important roles in energy homeostasis, glucose and lipid metabolism, reproduction, cardiovascular function, and immunity. They directly influence other organ systems, including the brain, liver, and skeletal muscle, and are significantly regulated by nutritional status. This newly discovered secretory function has extended the biological relevance of adipose tissue, which is no longer considered as only an energy storage site. SUMMARY: The functional roles, structures, synthesis, analytical aspects, and clinical significance of leptin, ghrelin, adiponectin, and resistin are summarized.

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.

Disulfide-dependent multimeric assembly of resistin family hormones.

Resistin, founding member of the resistin-like molecule (RELM) hormone family, is secreted selectively from adipocytes and induces liver-specific antagonism of insulin action, thus providing a potential molecular link between obesity and diabetes. Crystal structures of resistin and RELMbeta reveal an unusual multimeric structure. Each protomer comprises a carboxy-terminal disulfide-rich beta-sandwich "head" domain and an amino-terminal alpha-helical "tail" segment. The alpha-helical segments associate to form three-stranded coiled coils, and surface-exposed interchain disulfide linkages mediate the formation of tail-to-tail hexamers. Analysis of serum samples shows that resistin circulates in two distinct assembly states, likely corresponding to hexamers and trimers. Infusion of a resistin mutant, lacking the intertrimer disulfide bonds, in pancreatic-insulin clamp studies reveals substantially more potent effects on hepatic insulin sensitivity than those observed with wild-type resistin. This result suggests that processing of the intertrimer disulfide bonds may reflect an obligatory step toward activation.

Cloning and functional characterization of resistin-like molecule gamma.

Resistin, a recently discovered hormone that may play a crucial role in obesity-associated diabetes, is the founding member of a novel family of cysteine-rich proteins that are secreted by specific cell types. Three other members of this family have been described to date and were termed resistin-like molecules (RELMs). Here we describe the cloning and functional characterization of RELMgamma. The mouse RELMgamma-cDNA encodes a protein of 117 amino acids that contains a signal peptide leading to secretion of the protein. By Northern blotting the RELMgamma-mRNA is detectable in bone marrow, spleen, and lung as well as in peripheral blood granulocytes. Promyelocytic HL60 cells transfected with a RELMgamma expression plasmid have an increased proliferation rate compared to mock-transfected cells and display an altered response to retinoic acid-induced granulocytic differentiation. Taken together, these data provide the first experimental evidence that RELMgamma is a secreted molecule with a restricted expression pattern that may play a role in promyelocytic differentiation.

Dimerization of human recombinant resistin involves covalent and noncovalent interactions.

Resistin, an adipocyte secreted cysteine rich hormone has been implicated as molecular link between obesity and type 2 diabetes in a murine model. Although, at the protein level mouse and human resistin show remarkable similarities with respect to conserved cysteine residues, the physiological role of human resistin is not yet clear. In the present study we describe the purification and refolding of human recombinant resistin using two different refolding processes. Gel filtration analysis of protein refolded by both the methods revealed that human recombinant resistin, like mouse resistin, has a tendency to form dimers. Interestingly, dimerization of resistin appears to be mediated by both covalent (disulfide bond mediated) and non-covalent interactions as seen on reducing and non-reducing SDS-PAGE. Circular dichroism spectral analysis revealed that human resistin peptide backbone is a mixture of alpha-helical and beta-sheet conformation with significant amounts of unordered structure, similar to the mouse resistin. It is likely that the first cysteine (Cyst22) of human resistin, which is equivalent to mouse Cyst26, may be involved in stabilizing the dimers through covalent interaction.

Bacterial colonization leads to the colonic secretion of RELMbeta/FIZZ2, a novel goblet cell-specific protein.

BACKGROUND & AIMS: Goblet cells are highly polarized exocrine cells found throughout the small and large intestine that have a characteristic morphology due to the accumulation of apical secretory granules. These granules contain proteins that play important physiologic roles in cellular protection, barrier function, and proliferation. A limited number of intestinal goblet cell-specific proteins have been identified. In this study, we investigate the expression and regulation of RELMbeta, a novel colon-specific gene. METHODS: The regulation of RELMbeta messenger RNA expression was determined in LS174T, Caco-2, and HT-29 cell lines in response to stimulation with interleukin 13 and lipopolysaccharide. Quantitative reverse-transcription polymerase chain reaction, immunoblots, and immunohistochemistry were used to examine the expression of RELMbeta in BALB/c and C.B17.SCID mice housed in conventional, germ-free, and gnotobiotic environments. RESULTS: Messenger RNA for RELMbeta is restricted to the undifferentiated, proliferating colonic epithelium. Immunohistochemistry shows that this protein is expressed in goblet cells located primarily in the distal half of the colon and cecum with lower levels detectable in the proximal colon. High levels of RELMbeta can be detected in the stool of mice and humans, where it exists as a homodimer under nonreducing conditions. Interestingly, the secretion of RELMbeta is dramatically reduced in germ-free mice. Furthermore, introduction of germ-free mice into a conventional environment results in enhanced expression and robust secretion of RELMbeta within 48 hours. CONCLUSIONS: These studies define a new goblet cell-specific protein and provide the first evidence that colon-specific gene expression can be regulated by colonization with normal enteric bacteria.

Human recombinant resistin protein displays a tendency to aggregate by forming intermolecular disulfide linkages.

Resistin, a small cysteine rich protein secreted by adipocytes, has been proposed to be a link between obesity and type II diabetes by modulating the insulin signaling pathway and thus inducing insulin resistance. Resistin protein, with 11 cysteine residues, was not significantly homologous at the amino acid level to any other known cysteine rich proteins. Resistin cDNA derived from human subcutaneous adipose tissue was expressed in Escherichia coli as an N-terminal six-His-tag fusion protein. The overexpressed recombinant resistin was purified to homogeneity from inclusion bodies, after solubilization in 8 M urea, using a metal affinity column. While MALDI-TOF mass spectrometric analysis of the purified protein generated a single peak corresponding to the estimated size of 11.3 kDa, the protein exhibited a concentration-dependent oligomerization which is evident from size exclusion chromatography. The oligomeric structure was SDS-insensitive but beta-mercaptoethanol-sensitive, pointing to the importance of disulfide linkages in resistin oligomerization. Estimation of free cysteine residues using the NBD-Cl assay revealed a concentration- and time-dependent increase in the extent of formation of disulfide linkages. The presence of intermolecular disulfide bond(s), crucial in maintaining the global conformation of resistin, was further evident from fluorescence emission spectra. Circular dichroism spectra revealed that recombinant resistin has a tendency to reversibly convert from alpha-helical to beta-sheet structure as a direct function of protein concentration. Our novel observations on the biophysical and biochemical features of human resistin, particularly those shared with prion proteins, may have a bearing on its likely physiological function.

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.

Dimerization of resistin and resistin-like molecules is determined by a single cysteine.

Resistin is a peptide hormone secreted by adipocytes. Cysteine residues comprise 11 of 94 (12%) amino acids in resistin. The arrangement of these cysteines is unique to resistin and its recently discovered family of tissue-specific secreted proteins, which have been independently termed resistin-like molecules (RELMs) and the FIZZ (found in inflammatory zone) family. Here we show that resistin is a disulfide-linked homodimer that can be converted to a monomer by reducing conditions. The intestine-specific RELM beta has similar characteristics. Remarkably, however, the adipose-enriched RELM alpha is a monomer under non-reducing conditions. We note that RELM alpha lacks a cysteine residue, closest to the cleaved N terminus, that is present in resistin and RELM beta in multiple species. Conversion of this cysteine to alanine abolishes dimerization of resistin. Thus, a single disulfide bond is necessary to connect two resistin subunits in a homodimer. The additional 10 cysteines most likely participate in intramolecular disulfide bonds that define the conserved structure of the family members. The monomeric nature of RELM alpha suggests structural and potentially functional divergence between resistin and this close family member.

A family of tissue-specific resistin-like molecules.

We have identified a family of resistin-like molecules (RELMs) in rodents and humans. Resistin is a hormone produced by fat cells. RELMalpha is a secreted protein that has a restricted tissue distribution with highest levels in adipose tissue. Another family member, RELMbeta, is a secreted protein expressed only in the gastrointestinal tract, particularly the colon, in both mouse and human. RELMbeta gene expression is highest in proliferative epithelial cells and is markedly increased in tumors, suggesting a role in intestinal proliferation. Resistin and the RELMs share a cysteine composition and other signature features. Thus, the RELMs together with resistin comprise a class of tissue-specific signaling molecules.