Inhibition of adipocyte differentiation by resistin-like molecule alpha. Biochemical characterization of its oligomeric nature

A novel family of cysteine-rich secreted proteins with unique tissue distribution has recently been identified. One of the members, resistin (for "resistance to insulin"), also called FIZZ3, was identified in a screen for molecules that are down-regulated in mature adipocytes upon administration of thiazolidinediones. The prototypical member of this family was originally identified from bronchoalveolar lavage fluid of inflamed lungs and designated FIZZ1 ("found in inflammatory zone"). This molecule was also found to be highly expressed in adipose tissue and was named resistin-like molecule alpha (RELMalpha). Here we demonstrate that RELMalpha inhibits the differentiation of 3T3-L1 preadipocytes into adipocytes. RELMalpha has no effect on proliferation of 3T3-L1 preadipocytes. Pretreatment of 3T3-L1 preadipocytes with RELMalpha does not affect insulin- or platelet-derived growth factor-induced mitogenesis. IRS-1 phosphorylation and glucose transport stimulated by insulin in mature adipocytes were also unaffected by RELMalpha. We show that RELMalpha forms disulfide-linked homooligomers based on results from electrophoresis under reducing and nonreducing conditions, coimmunoprecipitation experiments as well as by mass spectrometry. In addition, RELMalpha is able to form heterooligomers with resistin but not RELMbeta. Since RELMalpha is expressed by adipose tissue and it is a secreted factor, our findings suggest that RELMalpha may be involved in the control of the adipogenesis as well as in the process of muscle differentiation.     

Identification of RELMgamma,a novel resistin-like molecule with a distinct expression pattern

We have identified RELMgamma, a novel member of the resistin-like molecule/found in inflammatory zone (RELM/FIZZ) family in mice and rats. Microarray and real-time RT-PCR experiments revealed a repression of RELMgamma mRNA in nasal respiratory epithelium of cigarette smoke-exposed versus untreated rats. The analysis of the physiological tissue-specific expression revealed highest expression in hematopoietic tissues, suggesting a cytokine-like role for RELMgamma. RELMgamma is most closely related to RELMalpha/FIZZ1. Despite the high similarity, the expression properties of the two genes are clearly distinct. While RELMgamma (approved symbol retnlg) is expressed in rat white adipose tissue, minute to no expression of RELMalpha was detected in that system. Thus, previous reports analyzing RELMalpha expression in rat adipose tissue might have been influenced by cross-hybridization with RELMgamma. Finally we could demonstrate that white adipose tissue of mice shows strong RELMalpha expression but only low levels of RELMgamma, indicating a species-specific gene regulation.     

嗜中性粒细胞是人抵抗素表达的主要细胞

抵抗素(resistin)是小鼠白色脂肪组织大量表达的富含半胱氨酸的 分泌型蛋白.近年研究发现,人与啮齿类动物的抵抗素组织表达分布存在很 大差异．小鼠抵抗素主要在白色脂肪组织表达,而人抵抗素主要在单核细 胞/巨噬细胞表达,且在骨髓组织中大量表达,但目前骨髓中的细胞定位还 不清楚．本研究的目的是明确成人骨髓及外周血白细胞中抵抗素表达细胞 的类型．免疫荧光法检测骨髓中抵抗素表达细胞,结果显示,抵抗素主要表 达在细胞核呈杆状和分叶核状的成熟粒细胞中,其中杆状核粒细胞表达较 高,分叶核粒细胞表达减弱．Anti-hresistin IgG-Biotin-PE单色荧光流 式细胞术分选外周血白细胞中抵抗素表达细胞后经瑞氏化学染色,结果显 示,抵抗素表达细胞主要为杆状和分叶核状的嗜中性粒细胞,还有少量嗜酸 性粒细胞,且抵抗素蛋白分布在细胞质中. RT-qPCR结果在RNA水平上证明, 人抵抗素在嗜中性粒细胞中大量表达．Anti-hresistin IgG-FITC和anti- HNL IgG-Biotin-PE 双色荧光流式细胞术进一步证明,抵抗素的主要表达细 胞为成熟的嗜中性粒细胞．嗜中性粒细胞在机体免疫防御中起重要作用, 人骨髓及外周血中抵抗素主要在成熟嗜中性粒细胞中表达,这一研究结论 为人抵抗素与炎症反应的关联性及其功能的进一步研究奠定了基础.     

Pref-1 and ADSF/resistin: two secreted factors inhibiting adipose tissue development.

Adipose tissue is the source of a wide array of factors of great biological significance that are involved in many aspects of organism physiology, including appetite control and peripheral metabolism. Here, we describe two secreted factors from adipose tissue that inhibit adipogenesis. Pref-1 is a preadipocyte secreted factor synthesized as a transmembrane protein that undergoes proteolitic cleavage to generate two distinct soluble forms. In vitro assays have demonstrated that only the large soluble form of Pref-1 is biologically active and inhibits adipocyte differentiation. In vivo, mice lacking Pref-1 expression show accelerated fat deposition, perinatal mortality and growth retardation as well as distinct skeletal malformations, highlighting the importance of Pref-1 during mouse development in addition to its role in adipose tissue development. ADSF/resistin is secreted by adipocytes and inhibits adipose cells differentiation in vitro. Its function is still unclear, but its expression and high circulating levels have been associated with an impairment of insulin action. The findings show that Pref-1 and possibly ADSF/resistin secretion control fat cell differentiation and adipose tissue development.     

Comparative studies of resistin expression and phylogenomics in human and mouse

Resistin is a newly identified adipocytokine that has been proposed to be a link between obesity and type 2 diabetes based on animal studies. However, the role of resistin in the pathogenesis of insulin resistance associated with obesity in humans remains unclear. We comparatively and quantitatively studied the tissue distributions of resistin mRNA between human and mouse. The expression level of resistin mRNA in human adipose tissue is extremely low but detectable by real-time PCR and is about 1/250 of that in the mouse. Remarkably, resistin mRNA is abundant in human primary acute leukemia cells and myeloid cell lines U937 and HL60, but not in the Raw264 mouse myeloid cell line. Resistin expression in U937 cells was not affected by lipopolysaccharide (LPS) or by ciglitazone, a PPARgamma ligand. Phylogenomics revealed that the human resistin gene is the ortholog of its murine counterpart and is located in a region of chromosome 19p13.3, which is syntenic to mouse chromosome 8A1. In addition to the resistin-like molecule (RELM) sequences already reported, bioinformatics analysis disclosed another RELM sequence in the vicinity of RELMbeta on human chromosome 3q13.1, but this sequence is unlikely to encode an expressed gene. Therefore, only two RELMs, resistin and RELMbeta, exist in humans, instead of the three RELMs, resistin, RELMalpha, and RELMbeta, that exist in mice. This finding provides a possible answer to the question of why only two RELMs have been cloned in humans and suggests that the RELM family is not well conserved in evolution and may function differently between species. Therefore, caution should be exercised in interpreting resistin as a link between obesity and insulin resistance in humans. The high expression of resistin in human leukemia cells suggests a hitherto unidentified biological function of resistin in leukocytes.     

Resistin overexpression impaired glucose tolerance in hepatocytes

Resistin is a 12.5-kDa cysteine-rich protein secreted from adipose tissue and is an important factor linking obesity with insulin resistance. Here, we investigated the effect of resistin on glucose tolerance in adult human hepatocytes (L-02 cells). In this study, resistin cDNA was transfected into L-02 cells, and glucose concentration and glucokinase activity were determined subsequently. The data indicated resistin impaired, insulin-stimulated glucose utilization, which implied liver was a target tissue of resistin. To understand its molecular mechanism, mRNA levels of key genes in glucose metabolism and insulin signaling pathway were analyzed. The results demonstrated resistin-stimulated expression of glucose-6-phosphatase (G6Pase), sterol regulatory element-binding protein 1c (SREBP1c) and suppressor of cytokine signaling 3 (SOCS-3), repressed expression of peroxisome proliferator-activated receptor gamma (PPARgamma) as well as insulin receptor substrate 2 (IRS-2). Given that glucokinase (GK) activity and glucose transporter 2 (GLUT2) expression were not altered, we presumed that resistin did not effect them. Moreover, resistin lowered mRNA levels of IRS-2 while stimulating SOCS-3 expression, which suggests it impairs glucose tolerance by blocking the insulin signal transduction pathway.     

Adipose tissue resistin expression is severely suppressed in obesity and stimulated by peroxisome proliferator-activated receptor gamma agonists

Elevated levels of the hormone resistin, which is secreted by fat cells, are proposed to cause insulin resistance and to serve as a link between obesity and type 2 diabetes. In this report we show that resistin expression is significantly decreased in the white adipose tissue of several different models of obesity including the ob/ob, db/db, tub/tub, and KKA(y) mice compared with their lean counterparts. Furthermore, in response to several different classes of antidiabetic peroxisome proliferator-activated receptor gamma agonists, adipose tissue resistin expression is increased in both ob/ob mice and Zucker diabetic fatty rats. These data demonstrate that experimental obesity in rodents is associated with severely defective resistin expression, and decreases in resistin expression are not required for the antidiabetic actions of peroxisome proliferator-activated receptor gamma agonists.     

Hemojuvelin: a new link between obesity and iron homeostasis

The adipose tissue may play an active role in systemic iron regulation and this role may be determinant in obese patients. Indeed, we reported previously that hepcidin, the iron-regulatory hormone, is expressed in adipose tissue and its messenger RNA (mRNA) expression is increased in adipose tissue of morbidly obese patients. The objectives of this study were to characterize the status of hemojuvelin (HJV), another iron-regulatory protein, within the adipose tissue of morbidly obese patients. Since cell-associated HJV acts as a coreceptor of bone morphogenetic protein (BMP) to enhance hepcidin expression in liver cells, we investigated the possible involvement of this pathway in adipose tissue in regulating hepcidin expression. HJV expression was studied in adipose tissue of morbidly obese patients. Soluble HJV blood concentrations were assessed. Hepcidin regulation through BMP pathway was investigated in cultured adipocytes. HJV was expressed both at mRNA and protein levels in adipose tissue. Moreover, its mRNA expression was highly increased in adipose tissue of obese patients and correlated with mRNA hepcidin expression levels. Interestingly, HJV expressed by adipose tissue may be effective since cultured adipocytes increased their hepcidin expression when challenged with BMP2 through Smad effectors. In addition, blood concentrations of soluble HJV were significantly increased. In conclusion, adipose tissue may influence iron homeostasis in obese patients by expressing major iron-regulatory proteins and the BMP signaling pathway could be involved in regulating hepcidin expression in this tissue.     

Decreased resistin expression in mice with different sensitivities to a high-fat diet.

The regulation of resistin, a new adipose-derived circulating factor, is the subject of controversy. In particular, the question of its modulation in obesity led to opposite results reported by two different groups. In the current study, we assayed adipocyte resistin mRNA using fluorescent real-time RT-PCR. We studied the expression of resistin in mice which are differently sensitive to diet-induced obesity: the FVB/n strain, which poorly responds to high-fat diet and transgenic mice that express human alpha 2A-AR in adipose tissue in the absence of beta 3-adrenergic receptor (AR) under the FVB genetic background which are highly sensitive to high-fat diet and develop hyperplastic obesity. We observed that FVB mice, which have no significant increased body weight after an 8-week high-fat diet period, exhibited no alteration of resistin expression. In contrast, the transgenic mice developing high-fat diet-induced obesity exhibited markedly downregulated adipocyte resistin mRNA. We also showed that obesity induced by gold thioglucose injection in FVB/n mice reduces the expression of resistin in isolated adipocytes. This argues for decreased expression of resistin as a hallmark of obesity. Moreover, our data show that feeding a high-fat diet is not a primary determinant of resistin regulation.