Induction of uncoupling protein-2 (UCP2) gene expression on the differentiation of rat preadipocytes to adipocytes in primary culture

We have examined uncoupling protein-2 (UCP2) gene expression in the adipose tissue of obese and normal rats and mice, and also in differentiated rat adipocytes in primary culture. Expression of the UCP2 gene was examined in rat and mouse adipose tissues using both RT-PCR and Northern blotting. Although the RT-PCR was not quantitative, the band corresponding to the UCP2 mRNA was stronger in white adipose tissue than in brown fat, regardless of the body weight of the rats. In agreement with the RT-PCR data, there was a higher level of UCP2 mRNA in the white adipocytes than in brown adipocytes, the level being greater in obese mice. Fibroblastic preadipocytes were obtained from the inguinal fat pad of suckling rats. Lipid droplets developed inside the cells upon differentiation and adipsin and UCP2 mRNAs were detected by Northern blotting. Both mRNAs were evident in the adipocytes at 4, 6, and 10 d after the induction of differentiation. There was no indication that the expression of UCP2 was markedly affected by the addition of leptin, dexamethasone or isoprenaline.     

Adipokine expression profile in adipocytes of different mouse models of obesity.

Adipose tissue produces and secretes multiple adipokines. Most studies on adipokine production/expression have been performed on whole adipose tissue. In addition, data concerning an overall of adipokine expression are scarce and can be heterogeneous depending on the obesity model studied. Our first aim was to compare the expression of adipokines involved in the interplay between obesity and insulin resistance in isolated adipocytes from different mouse models of obesity displaying different levels of weight gain and insulin sensitivity. The second aim was to determine perigonadal/subcutaneous ratio of each adipokine. Only resistin expression was decreased in obese mice without modifications in glucose and insulin blood levels. In addition to decreased levels of resistin, obesity models associated with hyperglycemia and hyperinsulinemia presented an increased expression of leptin and tumor necrosis factor-alpha (TNFalpha). Obese and diabetic mice were the only animals to exhibit high expression of plasminogen activator inhibitor type-1 and interleukin-6. All adipokines except TNFalpha were more heavily expressed in perigonadal than in subcutaneous adipocytes. Interestingly, fat-enriched diet and overweight on their own did not modify the distribution of adipokines between the two fat depots. However, severe obesity modified the distribution of proinflammatory adipokines. In conclusion, the level and number of adipokines with altered expression increased with obesity and hyperinsulinemia in mice. The physiopathological impact of depot-specific differences of adipokine expression in adipocytes remains to be clarified.     

Expression and secretion of inflammation-related adipokines by human adipocytes differentiated in culture: integrated response to TNF-alpha

The expression profile of a series of adipokine genes linked to inflammation has been examined by quantitative PCR during the differentiation of human preadipocytes to adipocytes in primary culture, together with the integrated effects of TNF-alpha on the expression of these adipokines in the differentiated adipocytes. Expression of the genes encoding adiponectin, leptin, and haptoglobin was highly differentiation dependent, the mRNA being undetectable predifferentiation with the level peaking 9-15 days postdifferentiation. Although angiotensinogen (AGT) and monocyte chemoattractant protein-1 (MCP-1) were both expressed before differentiation, the mRNA level increased markedly on differentiation. The expression of nerve growth factor (NGF) and plasminogen activator inhibitor-1 (PAI-1) fell after differentiation, whereas that of TNF-alpha and IL-6 changed little. Measurement of adiponectin, leptin, MCP-1, and NGF in the medium by ELISA showed that the protein secretion pattern paralleled cellular mRNA levels. Treatment of differentiated human adipocytes with TNF-alpha (5 or 100 ng/ml for 24 h) significantly decreased the level of adiponectin, AGT, and haptoglobin mRNA (by 2- to 4-fold), whereas that of leptin and PAI-1 was unchanged. In contrast, TNF-alpha induced substantial increases in IL-6, TNF-alpha, metallothionein, MCP-1, and NGF mRNAs, the largest increase being with MCP-1 (14.5-fold). MCP-1 and NGF secretion increased 8- to 10-fold with TNF-alpha, whereas leptin and adiponectin did not change. These results demonstrate that there are major quantitative changes in adipokine gene expression during differentiation of human adipocytes and that TNF-alpha has a pleiotropic effect on inflammation-related adipokine production, the synthesis of MCP-1 and NGF being highly induced by the cytokine.     

NGF gene expression and secretion in white adipose tissue: regulation in 3T3-L1 adipocytes by hormones and inflammatory cytokines

The sympathetic nervous system plays a central role in lipolysis and the production of leptin in white adipose tissue (WAT). In this study, we have examined whether nerve growth factor (NGF), a target-derived neurotropin that is a key signal in the development and survival of sympathetic neurons, is expressed and secreted by white adipocytes. NGF mRNA was detected by RT-PCR in the major WAT depots of mice (epididymal, perirenal, omental, mesenteric, subcutaneous) and in human fat (subcutaneous, omental). In mouse WAT, NGF expression was observed in mature adipocytes and in stromal vascular cells. NGF expression was also evident in 3T3-L1 cells before and after differentiation into adipocytes. NGF protein, measured by ELISA, was secreted from 3T3-L1 cells, release being higher before differentiation. Addition of the sympathetic agonists norepinephrine, isoprenaline, or BRL-37344 (beta(3)-agonist) led to falls in NGF gene expression and secretion by 3T3-L1 adipocytes, as did IL-6 and the PPARgamma agonist rosiglitazone. A substantial decrease in NGF expression and secretion occurred with dexamethasone. In contrast, LPS increased NGF mRNA levels and NGF secretion. A major increase in NGF mRNA level (9-fold) and NGF secretion (相似文献   

The gender- and fat depot-specific regulation of leptin, resistin and adiponectin genes expression by progesterone in rat

Progesterone affects lipid metabolism in adipose tissue and influences fat distribution in human. The aim of the study was to analyze the effect of progesterone on rat body and fat mass and on expression of genes encoding adipokines involved in the regulation of energy homeostasis. The results presented here indicate that progesterone administration to females caused increase in body and inguinal white adipose tissue mass. The increase of inguinal white adipose tissue mass is associated with the hypertrophy of adipocyte. The same dose of progesterone caused increase of its circulating concentration in males, however it barely reached the value observed in non-treated control females and did not have any effect on body and fat mass. The elevated circulating progesterone concentration was associated with an approximately 6- and 2-fold increase of leptin and resistin mRNA level respectively, and 2-fold decrease of adiponectin mRNA level only in inguinal white adipose tissue of females. RU 486, specific antagonist of progesterone receptor, abolished the effect of progesterone on the adipokine mRNA level in inguinal adipose tissue. In males, the elevated circulating progesterone concentration showed no effects on leptin, resistin or adiponectin mRNA level in inguinal, retroperitoneal or epididymal adipose tissue. Moreover, the results presented in this paper demonstrate a relatively high level of progesterone receptor mRNA in inguinal white adipose tissue of females, which was down-regulated in response to progesterone administration. In retroperitoneal adipose tissue of control females progesterone receptor mRNA level was approximately 3-fold lower as compared to inguinal adipose tissue. In inguinal, epididymal and retroperitoneal white adipose tissue of males progesterone receptor mRNA was hardly detected. Our results suggest that depot- and sex-dependent responsiveness of adipose tissue to the pharmacological dose of progesterone is controlled by both circulating concentration of progesterone and the white adipose tissue progesterone receptor level.     

Adipolin/C1qdc2/CTRP12 protein functions as an adipokine that improves glucose metabolism

Obesity is a major risk factor for the development of insulin resistance and type 2 diabetes. Adipose tissue secretes various bioactive molecules, referred to as adipokines, whose dysregulation can mediate changes in glucose homeostasis and inflammatory responses. Here, we identify C1qdc2/CTRP12 as an insulin-sensitizing adipokine that is abundantly expressed by fat tissues and designate this adipokine as adipolin (adipose-derived insulin-sensitizing factor). Adipolin expression in adipose tissue and plasma was reduced in rodent models of obesity. Adipolin expression was also decreased in cultured 3T3-L1 adipocytes by treatment with inducers of endoplasmic reticulum stress and inflammation. Systemic administration of adipolin ameliorated glucose intolerance and insulin resistance in diet-induced obese mice. Adipolin administration also reduced macrophage accumulation and proinflammatory gene expression in the adipose tissue of obese mice. Conditioned medium from adipolin-expressing cells diminished the expression of proinflammatory cytokines in response to stimulation with LPS or TNFα in cultured macrophages. These data suggest that adipolin functions as an anti-inflammatory adipokine that exerts beneficial actions on glucose metabolism. Therefore, adipolin represents a new target molecule for the treatment of insulin resistance and diabetes.     

Taurine chloramine modulates the expression of adipokines through inhibition of the STAT-3 signaling pathway in differentiated human adipocytes

To examine the possible role of taurine chloramine (TauCl) in modulating the expression of adipokines in adipose tissue associated with obesity, we evaluated the effect of TauCl in human differentiated adipocytes in response to IL-1β. To study the physiological effects of TauCl on adipokine expression, differentiated adipocytes were treated with IL-1β in the presence or absence of TauCl at concentrations ranging from 200 to 600 μM for 7 days. Cell culture supernatants and total RNA were analyzed by ELISA and real-time PCR, respectively, to determine protein and mRNA levels of adipokines, including adiponectin, leptin, IL-6, and IL-8. Levels of proteins involved in relevant signaling pathways were investigated by western blotting. Stimulation with IL-1β significantly decreased levels of adiponectin and leptin in adipocytes, but increased levels of IL-6 and IL-8 in a dose-dependent manner. Treatment with TauCl significantly reversed the modulation of adipokine expression by inhibiting STAT-3 signaling in IL-1β-stimulated adipocytes, independent of MAPK signaling. TauCl treatment more significantly modulated the expression of adipokines in adipocytes stimulated with IL-1β than that of non-stimulated adipocytes, suggesting that TauCl plays a significant role in modulating the expression of adipokines under inflammatory conditions. In conclusion, TauCl and other taurine derivatives that inhibit the STAT-3 signaling pathway can modulate expression of adipokines and thus may be useful as therapeutic agents for obesity-related diseases.     

White adipose tissue genome wide-expression profiling and adipocyte metabolic functions after soy protein consumption in rats

Obesity is associated with an increase in adipose tissue mass due to an imbalance between high dietary energy intake and low physical activity; however, the type of dietary protein may contribute to its development. The aim of the present work was to study the effect of soy protein versus casein on white adipose tissue genome profiling, and the metabolic functions of adipocytes in rats with diet-induced obesity. The results showed that rats fed a Soy Protein High-Fat (Soy HF) diet gained less weight and had lower serum leptin concentration than rats fed a Casein High-Fat (Cas HF) diet, despite similar energy intake. Histological studies indicated that rats fed the Soy HF diet had significantly smaller adipocytes than those fed the Cas HF diet, and this was associated with a lower triglyceride/DNA content. Fatty acid synthesis in isolated adipocytes was reduced by the amount of fat consumed but not by the type of protein ingested. Expression of genes of fatty acid oxidation increased in adipose tissue of rats fed Soy diets; microarray analysis revealed that Soy protein consumption modified the expression of 90 genes involved in metabolic functions and inflammatory response in adipose tissue. Network analysis showed that the expression of leptin was regulated by the type of dietary protein and it was identified as a central regulator of the expression of lipid metabolism genes in adipose tissue. Thus, soy maintains the size and metabolic functions of adipose tissue through biochemical adaptations, adipokine secretion, and global changes in gene expression.     

Zinc-alpha2-glycoprotein, a lipid mobilizing factor, is expressed and secreted by human (SGBS) adipocytes

Zinc-alpha2-glycoprotein (ZAG), a lipid mobilizing factor, is expressed in mouse adipose tissue and is markedly upregulated in mice with cancer cachexia. We have explored whether ZAG is expressed and secreted by human adipocytes, using SGBS cells, and examined the regulation of ZAG expression. ZAG mRNA was detected by RT-PCR in mature human adipocytes and in SGBS cells post-, but not pre-, differentiation to adipocytes. Relative ZAG mRNA levels increased rapidly after differentiation of SGBS cells, peaking at day 8 post-induction. ZAG protein was evident in differentiated adipocytes (by day 3) and also detected in the culture medium (by day 6) post-induction. The PPARgamma agonist rosiglitazone induced a 3-fold increase in ZAG mRNA level, while TNF-alpha led to a 4-fold decrease. Human adipocytes express and secrete ZAG, with ZAG expression being regulated particularly through TNF-alpha and the PPARgamma nuclear receptor. ZAG is a novel adipokine, which may be involved in the local regulation of adipose tissue function.     

Identification and validation of novel adipokines released from primary human adipocytes

Adipose tissue is a major endocrine organ, releasing signaling and mediator proteins, termed adipokines, via which adipose tissue communicates with other organs. Expansion of adipose tissue in obesity alters adipokine secretion, which may contribute to the development of metabolic diseases. Although recent profiling studies have identified numerous adipokines, the amount of overlap from these studies indicates that the adipokinome is still incompletely characterized. Therefore, we conducted a complementary protein profiling on concentrated conditioned medium derived from primary human adipocytes. SDS-PAGE/liquid chromatography-electrospray ionization tandem MS and two-dimensional SDS-PAGE/matrix-assisted laser desorption ionization/time of flight MS identified 347 proteins, 263 of which were predicted to be secreted. Fourty-four proteins were identified as novel adipokines. Furthermore, we validated the regulation and release of selected adipokines in primary human adipocytes and in serum and adipose tissue biopsies from morbidly obese patients and normal-weight controls. Validation experiments conducted for complement factor H, αB-crystallin, cartilage intermediate-layer protein, and heme oxygenase-1 show that the release and expression of these factors in adipocytes is regulated by differentiation and stimuli, which affect insulin sensitivity, as well as by obesity. Heme oxygenase-1 especially reveals to be a novel adipokine of interest. In vivo, circulating levels and adipose tissue expression of heme oxygenase-1 are significantly increased in obese subjects compared with lean controls. Collectively, our profiling study of the human adipokinome expands the list of adipokines and further highlights the pivotal role of adipokines in the regulation of multiple biological processes within adipose tissue and their potential dysregulation in obesity.     

Proteomic characterization of adipose tissue constituents, a necessary step for understanding adipose tissue complexity

The original concept of adipose tissue as an inert storage depot for the excess of energy has evolved over the last years and it is now considered as one of the most important organs regulating body homeostasis. This conceptual change has been supported by the demonstration that adipose tissue serves as a major endocrine organ, producing a wide variety of bioactive molecules, collectively termed adipokines, with endocrine, paracrine and autocrine activities. Adipose tissue is indeed a complex organ wherein mature adipocytes coexist with the various cell types comprising the stromal-vascular fraction (SVF), including preadipocytes, adipose-derived stem cells, perivascular cells, and blood cells. It is known that not only mature adipocytes but also the components of SVF produce adipokines. Furthermore, adipokine production, proliferative and metabolic activities and response to regulatory signals (i.e. insulin, catecholamines) differ between the different fat depots, which have been proposed to underlie their distinct association to specific diseases. Herein, we discuss the recent proteomic studies on adipose tissue focused on the analysis of the separate cellular components and their secretory products, with the aim of identifying the basic features and the contribution of each component to different adipose tissue-associated pathologies.     

Expression of Class III facilitative glucose transporter genes (GLUT-10 and GLUT-12) in mouse and human adipose tissues

We have examined whether GLUT-10 and GLUT-12, members of the Class III group of the recently expanded family of facilitative glucose transporters, are expressed in adipose tissues. The mouse GLUT-12 gene, located on chromosome 10, comprises at least five exons and encodes a 622 amino acid protein exhibiting 83% sequence identity and 91% sequence similarity to human GLUT-12. Expression of the GLUT-12 gene was evident in all the major mouse adipose tissue depots (epididymal, perirenal, mesenteric, omental, and subcutaneous white; interscapular brown). The GLUT-10 gene is also expressed in mouse adipose tissues and as with GLUT-12 expression occurred in the mature adipocytes as well as the stromal vascular cells. 3T3-L1 adipocytes express GLUT-10, but not GLUT-12, and expression of GLUT-12 was not induced by insulin or glucose. Both GLUT-10 and GLUT-12 expression was also found in human adipose tissue (subcutaneous and omental) and SGBS adipocytes. It is concluded that white fat expresses a wide range of facilitative glucose transporters.     

An immunohistochemical and in situ hybridisation study of the postnatal development of uncoupling protein-1 and uncoupling protein-1 mRNA in lamb perirenal adipose tissue

In the lamb, the uncoupling protein-1 (UCP1) content of perirenal adipose tissue at birth is an important factor in heat production by non-shivering thermogenesis and the prevention of hypothermia. This study examines UCP1 gene expression and protein content in perirenal adipose tissue over the first 15 days of life by in situ hybridisation and immunohistochemistry. UCP1 mRNA was detected at birth in 30% of adipocytes, and in approximately 24% of fat cells at 2 days of life. However, by 5 days of age and thereafter UCP1 mRNA was undetectable. Immunoreactive UCP1 was present in all adipocytes at birth and at 2 days of age, and remained detectable in a decreasing proportion of cells until day 10 of life. By 15 days of age no immunoreactive UCP1 was detected and the perirenal adipose tissue had the appearance of white fat. It is concluded that UCP1 gene expression is suppressed in most adipocytes in perirenal adipose tissue of newborn lambs, and gene expression rapidly falls in the remaining adipocytes over the first 5 days of postnatal life. In contrast, immunoreactive UCP1, a characteristic of brown adipose tissue, was present in many adipocytes for up to 10 days of age, suggesting that UCP1 has a long half-life in lambs. All adipocytes in perirenal adipose tissue of newborn lambs appear to be functionally brown, but over the first 2 weeks of postnatal life there is a complete transformation to white adipocytes.     

Appetite and energy balance signals from adipocytes

Interest in the biology of white adipose tissue has risen markedly with the recent surge in obesity and its associated disorders. The tissue is no longer viewed simply as a vehicle for lipid storage; instead, it is recognized as a major endocrine and secretory organ. White adipocytes release a multiplicity of protein hormones, signals and factors, termed adipokines, with an extensive range of physiological actions. Foremost among these various adipokines is the cytokine-like hormone, leptin, which is synthesized predominantly in white fat. Leptin plays a critical role in the control of appetite and energy balance, with mutations in the genes encoding the hormone or its receptor leading to profound obesity in both rodents and man. Leptin regulates appetite primarily through an interaction with hypothalamic neuroendocrine pathways, inhibiting orexigenic peptides such as neuropeptide Y and orexin A, and stimulating anorexigenic peptides such as proopiomelanocortin. White fat also secretes several putative appetite-related adipokines, which include interleukin-6 and adiponectin, but whether these are indeed significant signals in the regulation of food intake has not been established. Through leptin and the other adipokines it is evident that adipose tissue communicates extensively with other organs and plays a pervasive role in metabolic homeostasis.     

Thermogenic Ability of Uncoupling Protein 1 in Beige Adipocytes in Mice

Chronic adrenergic activation leads to the emergence of beige adipocytes in some depots of white adipose tissue in mice. Despite their morphological similarities to brown adipocytes and their expression of uncoupling protein 1 (UCP1), a thermogenic protein exclusively expressed in brown adipocytes, the beige adipocytes have a gene expression pattern distinct from that of brown adipocytes. However, it is unclear whether the thermogenic function of beige adipocytes is different from that of classical brown adipocytes existing in brown adipose tissue. To examine the thermogenic ability of UCP1 expressed in beige and brown adipocytes, the adipocytes were isolated from the fat depots of C57BL/6J mice housed at 24°C (control group) or 10°C (cold-acclimated group) for 3 weeks. Morphological and gene expression analyses revealed that the adipocytes isolated from brown adipose tissue of both the control and cold-acclimated groups consisted mainly of brown adipocytes. These brown adipocytes contained large amounts of UCP1 and increased their oxygen consumption when stimulated with norepinephirine. Adipocytes isolated from the perigonadal white adipose tissues of both groups and the inguinal white adipose tissue of the control group were white adipocytes that showed no increase in oxygen consumption after norepinephrine stimulation. Adipocytes isolated from the inguinal white adipose tissue of the cold-acclimated group were a mixture of white and beige adipocytes, which expressed UCP1 and increased their oxygen consumption in response to norepinephrine. The UCP1 content and thermogenic ability of beige adipocytes estimated on the basis of their abundance in the cell mixture were similar to those of brown adipocytes. These results revealed that the inducible beige adipocytes have potent thermogenic ability comparable to classical brown adipocytes.     

Regulation of metallothionein gene expression and secretion in rat adipocytes differentiated from preadipocytes in primary culture.

The gene encoding metallothionein, a low mol. wt. metal binding and stress response protein, is expressed in white adipose tissue. In the present study, metallothionein (MT-1) gene expression and factors regulating metallothionein production have been examined in adipocytes induced to differentiate from fibroblastic preadipocytes in primary cell culture. On the induction of differentiation, the metallothionein-1 gene was strongly expressed in the cells and metallothionein released into the medium. A peak in metallothionein-1 mRNA level and metallothionein secretion occurred at 2 and 10 days post-differentiation, respectively, with a decrease in protein release after this time. The metallothionein-1 gene was expressed in the adipocytes prior to the adipsin and lipoprotein lipase genes, suggesting that it is an early marker of adipocyte differentiation. The addition of the glucocorticoid, dexamethasone, led to a substantial increase in metallothionein-1 mRNA in the cells and metallothionein secretion. Insulin and leptin also stimulated metallothionein production, although the effect was small. Neither noradrenaline nor the beta3-adrenoceptor agonist, BRL 37 344, altered metallothionein release but forskolin and bromo-cAMP were stimulatory, markedly increasing both metallothionein-1 level and metallothionein secretion. It is suggested that metallothionein is a novel secretory product of the differentiated white adipocyte and that its production is regulated particularly by glucocorticoids and through a cAMP-dependent pathway.     

Adipokines identified as new downstream targets for adiponectin: lessons from adiponectin-overexpressing or -deficient mice

Adipokines play a central role in the pathogenesis of the metabolic syndrome. Among them, adiponectin (ApN), a master regulator of immune and fuel homeostasis, is decreased. Identifying downstream adipokines targeted by ApN may help in deciphering this syndrome. We have generated transgenic mice, allowing persistent and moderate overexpression of ApN (ApN-Overex) specifically in white adipose tissue (AT). We took advantage of this model to unravel the adipokine secretion profile triggered by ApN. AT was fractionated into adipocytes and stromal-vascular cells (SVC), which were cultured for 8 h. Profiling of secretory products by antibody arrays and subsequent ELISAs showed that the secretion of three proinflammatory factors (IL-17B, IL-21, TNFα) and three hematopoietic growth factors [GF; thrombopoietin and granulocyte (macrophage) colony-stimulating-factors] was reduced in adipocytes of ApN-Overex mice compared with wild-type mice. In the SVC of these mice, besides the hematopoietic GFs, the secretion of another GF (vascular endothelial GF receptor 1), two chemokines (RANTES and ICAM-1), and two proinflammatory factors (IL-6 and IL-12p70) was reduced as well. Only one cytokine, IL-1 receptor 4, was oversecreted by SVC of ApN-Overex mice, which may exhibit anti-inflammatory properties. Most of these changes in secretion were due to corresponding changes in mRNAs. A reverse profile of adipokine expression was observed in ApN-KO mice. In conclusion, ApN regulates in vivo the secretion of downstream adipokines, thereby inducing a shift of the immune balance in both adipocytes and SVC toward a less inflammatory phenotype. These downstream adipokines may be new therapeutic targets for the management of the metabolic syndrome.