Downregulation of FLIP by cycloheximide sensitizes human fat cells to CD95-induced apoptosis

Adipocyte apoptosis is an important regulator of adipocyte number in fat depots. We have previously shown that an inhibition of protein synthesis sensitizes human adipocytes for apoptosis. In vivo, dramatic changes in the fat cell's protein expression should be anticipated under special conditions such as calorie restriction. Here, we studied the underlying mechanism by which human preadipocytes and adipocytes are sensitized for death receptor induced apoptosis in vitro.The protein synthesis blocker cycloheximide (CHX) sensitized human fat cells for CD95-induced apoptosis in a caspase-dependent manner. Treatment with CHX differentially changed expression of pro- and anti-apoptotic proteins. Most noticeably, FLICE-like inhibitory protein (FLIP) expression rapidly decreased during CHX treatment. Reduction of FLIP levels resulted in undetectable amounts of FLIP at the CD95 death-inducing signaling complex (DISC) upon CD95 stimulation, thereby enhancing recruitment and activation at caspase-8. Down-regulation of FLIP by shRNA sensitized preadipocytes for CD95-induced apoptosis. In mice, adipose tissue mRNA levels of Flip were down-regulated upon fasting.In conclusion, we identify FLIP as an important regulator of apoptosis sensitivity in fat cells. Modulating adipocyte homeostasis by apoptosis might provide a new therapeutic concept to get rid of excess adipose tissue, and FLIP might be a possible target molecule.     

URB is abundantly expressed in adipose tissue and dysregulated in obesity

Dysregulated production of adipocytokines in obesity is involved in the development of metabolic syndrome. URB/DRO1 contains N-terminal signal sequence and is thought to play a role in apoptosis of tumor cells. In the present study, we investigated the expression pattern of URB mRNA in adipose tissue and secretion from cultured adipocytes. In human and mouse, URB mRNA was predominantly expressed in adipose tissue and was downregulated in obese mouse models, such as ob/ob, KKAy, and diet-induced obese mice. In 3T3L1 adipocytes, insulin, TNF-α, H₂O₂ and hypoxia decreased URB mRNA level. This regulation was similar to that for adiponectin and opposite to MCP-1. URB protein was secreted in media of URB cDNA-stably transfected cells and endogenous URB was detected in media of cultured human adipocytes. In conclusion, the expression pattern of URB suggests its role in obesity and the results suggest that URB is secreted, at least in part, from adipocytes.     

Immortal brown adipocytes from p53-knockout mice: differentiation and expression of uncoupling proteins

Brown adipose tissue (BAT) is the specific site for metabolic heat production in mammals. To establish a novel immortal brown adipocyte cell line, the stromal-vascular fraction containing preadipocytes was obtained from interscapular BAT of mice deficient of a tumor-suppressor gene p53. The p53-deficient cells, tentatively named as HB2 cells, could be cultured in vitro after repeated passages and differentiated into adipocytes in the presence of insulin, T3 and/or troglitazone, expressing some adipocyte-specific genes and accumulating intracellular lipid droplets. The mRNA level of uncoupling protein 1 (UCP1), a mitochondrial protein specifically present in brown adipocytes, was undetectable in HB2 preadipocytes, but increased after adipose differentiation. In HB2 adipocytes, UCP1 mRNA expression was markedly activated after stimulation of the beta-adrenergic receptor pathway. The mRNA of UCP2 and UCP3, recently cloned isoforms of UCP1, were also detected in HB2 adipocytes, but their levels were not influenced by adrenergic stimulation. Thus HB2 cells seem useful for in vitro studies of BAT and UCP functions.     

Decreased Expression Of apM1 in Omental and Subcutaneous Adipose Tissue of Humans With Type 2 Diabetes

We have screened a subtracted cDNA library in order to identify differentially expressed genes in omental adipose tissue of human patients with Type 2 diabetes. One clone (#1738) showed a marked reduction in omental adipose tissue from patients with Type 2 diabetes. Sequencing and BLAST analysis revealed clone #1738 was the adipocyte-specific secreted protein gene apM1 (synonyms ACRP30, AdipoQ, GBP28). Consistent with the murine orthologue, apM1 mRNA was expressed in cultured human adipocytes and not in preadipocytes. Using RT-PCR we confirmed that apM1 mRNA levels were significantly reduced in omental adipose tissue of obese patients with Type 2 diabetes compared with lean and obese normoglycemic subjects. Although less pronounced, apM1 mRNA levels were reduced in subcutaneous adipose tissue of Type 2 diabetic patients. Whereas the biological function of apM1 is presently unknown, the tissue specific expression, structural similarities to TNFα and the dysregulated expression observed in obese Type 2 diabetic patients suggest that this factor may play a role in the pathogenesis of insulin resistance and Type 2 diabetes.     

猪脂肪组织和原代脂肪细胞中GPR43基因的转录表达规律

根据GenBank已发表的人、小鼠及大鼠GPR43(G protein-coupled receptor 43)基因序列, 设计并合成一对引物, RT-PCR扩增获得猪GPR43基因cDNA, 并利用PCR技术检测该基因在不同猪种、不同发育阶段、不同部位脂肪组织及原代脂肪细胞中的转录表达规律。结果显示, 成功克隆猪GPR43 cDNA片段, 长度为486 bp (GenBank登陆号为EU122439); 同源性分析发现, 猪GPR43与人、小鼠和大鼠同源性达83%以上; GPR43 mRNA表达量在脂肪型猪种上显著高于瘦肉型猪种, 随月龄增长表达量逐渐上升, 且皮下脂肪表达量较内脏脂肪高; 在猪前体脂肪细胞诱导分化过程中, GPR43 mRNA表达量呈时间依赖性升高。揭示GPR43 mRNA表达与猪肥胖程度、年龄、脂肪沉积部位以及脂肪细胞分化程度密切相关。     

Identification of omentin as a novel depot-specific adipokine in human adipose tissue: possible role in modulating insulin action

Central (visceral) obesity is more closely associated with insulin resistance, type 2 diabetes, and cardiovascular disease than is peripheral [subcutaneous (sc)] obesity, but the underlying mechanism for this pathophysiological difference is largely unknown. To understand the molecular basis of this difference, we sequenced 10,437 expressed sequence tags (ESTs) from a human omental fat cDNA library and discovered a novel visceral fat depot-specific secretory protein, which we have named omentin. Omentin ESTs were more abundant than many known adipose genes, such as perilipin, adiponectin, and leptin in the cDNA library. Protein sequence analysis indicated that omentin mRNA encodes a peptide of 313 amino acids, containing a secretory signal sequence and a fibrinogen-related domain. Northern analysis demonstrated that omentin mRNA was predominantly expressed in visceral adipose tissue and was barely detectable in sc fat depots in humans and rhesus monkeys. Quantative real-time PCR showed that omentin mRNA was expressed in stromal vascular cells, but not fat cells, isolated from omental adipose tissue, with >150-fold less in sc cell fractions. Accordingly, omentin protein was secreted into the culture medium of omental, but not sc, fat explants. Omentin was detectable in human serum by Western blot analysis. Addition of recombinant omentin in vitro did not affect basal but enhanced insulin-stimulated glucose uptake in both sc (47%, n = 9, P = 0.003) and omental (approximately 30%, n = 3, P < 0.05) human adipocytes. Omentin increased Akt phosphorylation in the absence and presence of insulin. In conclusion, omentin is a new adipokine that is expressed in omental adipose tissue in humans and may regulate insulin action.     

Expression of angiotensin II receptors type 1 and type 2 in human preadipose cells during differentiation.

Human adipose tissue expresses all the components necessary for the production of angiotensin peptides. Although local effects of angiotensin II on cells from adipose tissue are beginning to be recognised, the expression of angiotensin receptors on human preadipocytes and adipocytes is still controversial. This study addresses the issue by monitoring the mRNA levels as well as the protein production of angiotensin II receptors of type 1 and 2 (AT 1 and AT 2 ) during differentiation of primary human preadipocytes in culture and in mature adipocytes. mRNA levels of the two receptor types are inversely correlated during adipose conversion. AT 1 receptor mRNA is greatly diminished within 12 days after induction of differentiation, while AT 2 receptor mRNA is elevated. mRNA levels of mature adipocytes confirm this trend. The regulation is not seen as strongly on the protein level. The amount of AT 2 receptor protein is increased, correlating well with the rise in specific glycerol-3-phosphate dehydrogenase activity of the cells, but the AT 1 receptor protein does not vary during the whole differentiation period. As the functional role of AT 2 receptors in adipose tissue is not known to date, further studies have to show if the AT 1 -mediated inhibitory actions on adipose conversion are downregulated in differentiating cells through decreased AT 1 /AT 2 receptor ratio.     

Regulation of lipoprotein lipase activity and mRNA content in rat epididymal adipose tissue in vitro by recombinant tumour necrosis factor.

Tumour necrosis factor (TNF) has previously been shown to decrease lipoprotein lipase (LPL) activity and mRNA levels in 3T3-L1 cells and in adipose tissue from rats and guinea pigs when injected in vivo, but not to alter LPL activity in human adipocytes incubated in vitro. The effect of recombinant human TNF on LPL activity and mRNA levels in rat epididymal adipose tissue incubated in vitro was examined. LPL activity and mRNA levels fell in adipose tissue taken from fed rats and incubated in Krebs-Henseleit bicarbonate medium with glucose. The addition of insulin and dexamethasone prevented these falls. TNF (400 ng/ml) produced a fall of approx. 50% in LPL activity after 2 h of incubation and of approx. 30% in LPL mRNA levels after 3 h. TNF did not decrease LPL activity in isolated adipocytes. These results demonstrate that rat adipose tissue incubated in vitro is responsive to TNF whereas isolated adipocytes are not.     

Expression of the adrenomedullin gene in adipose tissue

Adrenomedullin (AM) is a potent vasodilating peptide originally isolated from human pheochromocytoma cells. This report concerns the expression and secretion of AM from adipose tissue. Northern blot analysis demonstrated marked expression of AM mRNA in mouse adipose tissue. Expression levels in adipose tissues were 2.5-3.2 times higher than in the kidney. AM mRNA level in mature adipocytes was 7.3 times higher than in the stroma-vascular fraction of adipose tissue. In mature adipocyte culture, time-dependent increase of AM peptide concentration in the culture medium was detected. AM expression was also detected in human subcutaneous adipose tissue. Adipose AM expression significantly increased in obesity mouse model, high-fat diet fed mice and ob/ob mice. These results suggest that adipose tissue, especially mature adipocytes, is major source of AM in the body, and that adipocyte-derived AM plays a pathophysiological role in obesity.     

Metallothionein gene expression in human adipose tissue from lean and obese subjects.

Expression of the gene encoding metallothionein, a low molecular-weight cysteine-rich, stress-response and metal-binding protein was examined in human adipose tissue. The mRNA for MT-2A, a major metallothionein isoform in humans, was detected in subcutaneous fat using a specific antisense oligonucleotide probe. The level of MT-2A mRNA was significantly higher in a group of obese subjects than in a lean group, paralleling a similar increase in ob mRNA. A two-week period on a diet of 800 calories/day did not lead to any significant change in MT-2 mRNA levels. Separation of mature adipocytes from the cells of the stromal vascular fraction indicated that in human adipose tissue the metallothionein (MT-2A) gene is expressed both in adipocytes and in other cells of the tissue.     

The regulation of adipogenesis through GPR120

Recently, it has been found that long-chain fatty acids activate the G protein-coupled receptors (GPRs), GPR120 and GPR40. However, there have been no reports to date on the possible physiological roles of these GPRs in adipose tissue development and adipocyte differentiation. GPR120 mRNA was highly expressed in the four different adipose tissues, and the amount of mRNA was elevated in adipose tissues of mice fed a high fat diet. However, GPR40 mRNA was not detected in any of the adipose tissues. The expression of GPR120 mRNA was higher in adipocytes compared to stromal-vascular (S-V) cells. The level of GPR120 mRNA increased during adipocyte differentiation in 3T3-L1 cells. Similar results were observed in human adipose tissue, human preadipocytes, and cultured adipocytes. Moreover, use of a small interference RNA (siRNA) to down-regulate GPR120 expression resulted in inhibition of adipocyte differentiation. Our results suggest that GPR120 regulates adipogenic processes such as adipocyte development and differentiation.     

Ligand activation of peroxisome proliferator-activated receptor gamma induces apoptosis of leukemia cells by down-regulating the c-myc gene expression via blockade of the Tcf-4 activity

The peroxisome proliferator-activated receptor gamma (PPAR gamma), a member of the nuclear receptor superfamily, is expressed at highest levels in adipose tissue and functions as a central regulator in the process of adipocyte differentiation. In the present study, we showed that human leukemic cell lines, not only myeloid but also lymphoid, express PPAR gamma and its activation by natural ligand (15-deoxy-Delta(12,14) - prostaglandin J(2)) and synthetic ligand (troglitazone) profoundly inhibited their proliferation by induction of apoptosis preferentially in the serum-free culture. We pursued its mechanism using the representative cell lines, and found that induction of apoptosis was accompanied by caspase-3 activation and specifically blocked by its inhibitor. While status of several apoptosis-related molecules remained unchanged, the c-Myc expression was markedly down-regulated within 24 h after troglitazone treatment. The c-myc mRNA levels were dramatically reduced at 1 h and became undetectable at 12 h after troglitazone treatment, which proved to be accompanied by complete blockade of the Tcf-4 activity in the electrophoretic mobility shift assay. We succeeded in establishing HL-60 cell lines growing well in the presence of troglitazone in the long-term serum-free culture. They showed neither induction of apoptosis nor down-regulation of the c-Myc expression via blockade of the Tcf-4 activity after troglitazone treatment. This is the first identification of the linkage between PPAR gamma-mediated apoptosis and down-regulation of the c-myc gene expression.     

Galectin-1 induces cell adhesion to the extracellular matrix and apoptosis of non-adherent human colon cancer Colo201 cells

To isolate cDNAs for molecules involved in cell adhesion to the extracellular matrix, expression cloning with non-adherent colon cancer Colo201 cells was carried out. Four positive clones were isolated and, when sequenced, one was found to be galectin-1, a beta-galactoside-binding protein. When cultured on fibronectin-, laminin-, and collagen-coated and non-coated dishes, the adherent galectin-1 cDNA-transfected Colo201 cells increased and spread somewhat. Immunofluorescence staining revealed that galectin-1 was expressed inside and outside of Colo201 cells. The adhesion was dependent on the carbohydrate-recognition domain of galectin-1 since lactose inhibited the adhesion and exogenously-added galectin-1 caused the adhesion. PD58059, an inhibitor of mitogen-activated protein kinase, or LY294002, a phosphoinositide 3-OH kinase inhibitor, decreased the adhesion. Furthermore, the expression of galectin-1 in Colo201 cells induced apoptotic cell death, while exogenously-added galectin-1 did not cause apoptosis. These results indicate that galectin-1 plays a role in both cell-matrix interactions and the inhibition of Colo201 cell proliferation, and suggest that galectin-1 expressed in cells could be associated with apoptosis.     

Molecular characterization of a tandem-repeat galectin-9 (RuGlec9) from Korean rose bitterling (Rhodeus uyekii)

Galectin-9 is a b-galactoside-binding lectin that regulates many cellular functions, ranging from cell adhesion to pathogen recognition. We isolated and characterized the cDNA of tandem-repeat galectin-9 (RuGlec9) from the Korean rose bitterling (Rhodeus uyekii), an endemic Korean fish belonging to the Acheilognathinae subfamily of the Cyprinidae family. RuGlec9 cDNA is 1486 bp long and encodes a polypeptide of 323 amino acids containing two carbohydrate-recognition domains connected by a linker peptide. The deduced amino acid sequence of RuGlec9 shows 45-84% amino acid sequence identity to other galectin-9 sequences, including those from mammals and fish. RuGlec9 appeared in a large cluster with other galectin-9 sequences from fish and is more closely related to galectin-9 from Danio rerio than to those of other fish and mammals. RuGlec9 mRNA was expressed highly in the testis, spleen, intestine, stomach, and liver, and moderately in the brain, kidney, ovary, and gills of normal Korean rose bitterling. RuGlec9 mRNA expression in the spleen was increased by lipopolysaccharide. These results suggest that RuGlec9 plays a role in innate immunity in Korean rose bitterling.     

The effect of the HIV protease inhibitor ritonavir on proliferation, differentiation, lipogenesis, gene expression and apoptosis of human preadipocytes and adipocytes.

HIV patients in highly active antiretroviral therapy (HAART) develop lipodystrophy and insulin resistance. Protease inhibitors have been shown to alter adipocyte metabolism in murine cell lines. In this study, biological effects of the HIV protease inhibitor, ritonavir, were investigated on human SGBS preadipocytes and adipocytes. Ritonavir dose-dependently impaired preadipocyte proliferation and adipogenic differentiation. Gene expression analysis measured by real-time PCR, showed no effect of ritonavir (up to 20 microM) on expression of mRNA of PPARgamma2 and SREBP1c, but suppressed adiponectin mRNA while increasing IL-6 mRNA expression. In human adipocytes, ritonavir at therapeutic concentrations inhibited insulin-stimulated lipogenesis, reduced GLUT4 mRNA, fatty acid synthase and adiponectin expression, while increasing IL-6 mRNA expression. Finally, long-term treatment (72 and 120 h) of SGBS adipocytes but not preadipocytes with ritonavir induced apoptosis in up to 15% of the cells. All together, these data show effects of ritonavir on human preadipocytes and adipocytes aiming at reducing adipose tissue mass and increasing insulin resistance. These in vitro findings may partly explain the clinical findings in patients under HAART. Furthermore, SGBS cells may serve as a useful tool in further investigation of the mechanism of protease inhibitor action in human adipocytes.     

TSH receptor in adipose cells.

Although there have been reports supporting the presence of the TSH receptor (TSHR) in human adipose tissue, these findings are still not universally accepted. Contributing to the controversy is a paucity of data about the physiological role the TSH receptor might play in adipose cells. In addition to mature lipid-filled adipocytes, adipose tissue also harbors a pool of specialized, fibroblast-like preadipocytes within the stromal-vascular compartment. Upon appropriate induction, preadipocytes can either differentiate into adipocytes or undergo apoptosis. Since TSHR has been detected in preadipocytes and adipocytes, its potential impact on adipose tissue function may relate to differentiation stage-specific cellular properties.     

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.