A Human-Specific Role of Cell Death-Inducing DFFA (DNA Fragmentation Factor-)-Like Effector A (CIDEA) in Adipocyte Lipolysis and Obesity
http://www.100md.com
糖尿病学杂志 2005年第6期
1 Department of Medicine, Karolinska Institutet, Karolinska University Hospital Huddinge, Stockholm, Sweden
2 Wenner-Gren Institute, Stockholm University, Stockholm, Sweden
3 Department of Surgery, Karolinska Institutet, Karolinska University Hospital Huddinge, Stockholm, Sweden
ABSTRACT
Elevated circulating fatty acid concentration is a hallmark of insulin resistance and is at least in part attributed to the action of adipose tissue-derived tumor necrosis factor- (TNF-) on lipolysis. Cell death-inducing DFFA (DNA fragmentation factor-)-like effector A (CIDEA) belongs to a family of proapoptotic proteins that has five known members in humans and mice. The action of CIDEA is unknown, but CIDEA-null mice are resistant to obesity and diabetes. We investigated CIDEA in adipose tissue of obese and lean humans and mice. The mRNA was expressed in white human fat cells and in brown mouse adipocytes. The adipose mRNA expression of CIDEA in mice was not influenced by obesity. However, CIDEA expression was decreased twofold in obese humans and normalized after weight reduction. Low adipose CIDEA expression was associated with several features of the metabolic syndrome. Human adipocyte depletion of CIDEA by RNA interference stimulated lipolysis and increased TNF- secretion by a posttranscriptional effect. Conversely, TNF- treatment decreased adipocyte CIDEA expression via the mitogen-activated protein kinase c-Jun NH2-terminal kinase. We propose an important and human-specific role for CIDEA in lipolysis regulation and metabolic complications of obesity, which is at least in part mediated by cross-talk between CIDEA and TNF-.
Obesity is an excess of body fat that develops from a combination of genetic and environmental factors, leading to a disturbed balance between energy intake and energy expenditure (1). The prevalence is increasing in both developed and developing countries and has reached epidemic proportions (2,3). Obesity, in particular abdominal obesity, is closely associated with insulin resistance, non-insulin-dependent diabetes (type 2 diabetes), dyslipidemia, and cardiovascular disease (4).
Elevated circulating fatty acid levels in obesity resulting from increased basal adipocyte lipolysis could cause perturbations such as insulin resistance and type 2 diabetes (5). The exact molecular mechanisms causing activation of basal lipolysis are so far unknown (6), although the cytokine tumor necrosis factor- (TNF-) is a potential candidate. The production of adipose tissue-derived TNF- is increased in human obesity (7), and TNF- stimulates adipocyte lipolysis in rodent (8,9) and human fat cells (10,11).
The mammalian gene cell death-inducing DFFA (DNA fragmentation factor-)-like effector A (CIDEA) was recently identified and characterized (12). CIDEA belongs to a family of proapoptotic proteins (12) that has five known members in the transcriptomes of humans and mice (13). CIDEA is highly homologous to FSP27 (12), an adipose-specific gene whose expression is associated with terminal differentiation of fat cells (14). The expression of FSP27 (also called clone 47) is regulated by the tumor necrosis pathway (15).
Although the murine expression pattern of CIDEA has been studied and CIDEA-null mice are resistant to diet-induced obesity and diabetes (16), little else is known regarding the function of the CIDEA gene. CIDEA transcripts of various lengths are present in different human tissues (e.g., heart, skeletal muscle, brain, placenta, and kidney) (12), but there are no reports about CIDEA in human adipose tissue. Preliminary results from microarray studies in our laboratory demonstrated that CIDEA is significantly expressed in human white adipose tissue (WAT). This is in contrast to mice, where CIDEA is expressed in brown adipose tissue (BAT) but not detectable in WAT (16). We investigated the role of this gene in human WAT and obesity and show a specific role for CIDEA in regulating lipolysis in white human fat cells. We found interactions between CIDEA and TNF- that may be linked to the insulin-resistant phenotype of many obese subjects.
RESEARCH DESIGN AND METHODS
One cohort consisted of 40 men and 146 women, all healthy and free of medication but with a large interindividual range in BMI (18eC50 kg/m2). The subjects were divided into an obese (n = 143, BMI >30 kg/m2) and a nonobese (n = 43) subgroup. Twelve of the obese healthy subjects (BMI 35eC50 kg/m2) underwent gastric banding to treat their obesity. Fat tissue samples were obtained before and 2eC4 years after surgery in a weight-stable condition. These subjects were part of a larger cohort described previously (17). All subjects were examined at 8 A.M. after an overnight fast. Their height, weight, and waist circumference were measured. Venous plasma samples were drawn for the analysis of insulin and glucose and calculation of the homeostasis model assessment (HOMA) index ([plasma glucose (mmol/l) x plasma insulin (mU/l)]/22.5) (18), an indirect measure of in vivo insulin sensitivity. Thereafter, needle biopsies of subcutaneous adipose tissue (1eC2 g) were obtained under local anesthesia. For experimental studies, subcutaneous adipose tissue was obtained from healthy subjects after elective cosmetic liposuction. The studies were approved by the ethical committee at Karolinska Institutet and explained in detail to each of the subjects, from whom informed consent was obtained.
Mice.
A total of 18 male 7-week-old mice of three different strains (NMRI, C57BL/6J, and 129Sv/Pas, n = 6 from each strain; Scanbur B&K Universal, Sollentuna, Sweden) were fed normal pelleted chow (R70 Standard Diet; Lactamin, Vadstena, Sweden) with or without the addition of cafeteria diet ad libitum for 15 weeks. The mice had free access to water and were kept on a 12/12 h light/dark cycle. They were then killed by carbon dioxide anesthesia followed by cervical dislocation. The epididymal fat pads and interscapular BAT were dissected and snap frozen in liquid nitrogen for subsequent RNA isolation. All animal experimentation was performed in accordance with institutional guidelines, and ethical permission was obtained from the Northern Stockholm animal ethics committee.
Human adipose tissue and preadipocyte culture.
Adipose tissue (1 g) was immediately frozen in liquid nitrogen for subsequent RNA analysis. Fresh adipose tissue pieces were incubated as described for 2 h (19). Glycerol release to the medium was determined and related to the number of incubated fat cells, as previously described (20). Glycerol release is linear with incubation time for at least 4 h (21). Tissue was collagenase treated and mean fat cell size and mean fat cell weight determined as previously described (22). Preadipocytes were isolated from the stromal fraction of adipose tissue and differentiated into adipocytes as previously described (23), reaching full differentiation after 12eC14 days. The cells were seeded out in 12-well plates at a density of 30,000 cells/cm2. For studies of TNF--induced CIDEA downregulation, preadipocytes were incubated in the presence of 100 ng/ml TNF- in combination with different concentrations of the specific mitogen-activated protein kinase (MAPK) inhibitors SP600125 (for c-Jun NH2-terminal kinase [JNK]), PD98059 (for p44/42, also termed extracellular signal-regulated kinase 1/2), and SB203580 (for stress/cytokine-activated kinase, or p38) (all from Sigma, St Louis, MO) as described before (11,24). The MAPK cascades activate different transcription factors that mediate TNF--regulated gene expression (24). TNF- was added on day 10 (1 h after addition of the inhibitors) or on day 12 (alone), and the cells were incubated for 48 h before RNA isolation. Control cells were incubated in medium alone. Lipolysis in preadipocytes was assessed as glycerol release into the medium, as previously described (20).
RNA interference and protein analysis.
Optimal transfection conditions of human preadipocytes were initially determined by titrating different amounts of fluorescent short interfering RNA (siRNA) duplexes and the transfection reagent RNAiFect (both from Qiagen, Hilden, Germany). Analysis by fluorescence microscopy 4eC12 h later showed a transfection efficiency ranging from 5 to 70%, depending on the different conditions. These experiments served as a basis for optimizing CIDEA gene silencing. Cells at day 8 of differentiation (when the cells have developed lipid droplets but are not fully differentiated) were transfected with or without 1 or 2 e of CIDEA siRNA (Qiagen, Hilden, Germany) and incubated for 24 h, a time point where a significant gene silencing effect was observed. Cells incubated without siRNA served as controls. To control for nonspecific gene silencing effects, parallel cells were transfected with siRNA without known similarities to human sequences or with CIDEA siRNA without RNAiFect. Conditioned cell media aliquots were then analyzed for glycerol content while cells were lysed for RNA or protein isolation. Perilipin protein levels were detected by Western blot and related to -actin protein levels, as previously described (11). Protein release into the cell media was determined by a radioimmunoassay for adiponectin (Linco, St Charles, MO) and with Quantikine human immunoassays (R&D Systems, Abingdon, U.K.) for TNF- and monocyte chemoattractant protein-1 (MCP-1). The kits were used according to instructions from the manufacturers.
mRNA quantitation.
Total RNA was extracted from human or mouse adipose tissue, differentiated preadipocytes, or isolated adipocytes and reverse-transcribed as previously described (25). Quantitative real-time PCR was performed in an iCycler IQ (Bio-Rad Laboratories, Hercules, CA), using the less expensive SYBR Green-based technology for CIDEA quantitation in large cohorts (human and mouse) and mRNA levels of perilipin (PLIN), MCP-1, and the reference genes -2 microglobulin (2-MG; human and mouse) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). TaqMan probes (Applied Biosystems, Foster City, CA) were used for CIDEA and TNF- measurements in smaller experiments. 2-MG was used as a reference gene except for the TNF--treated samples, where 2-MG in our hands appears to be regulated by TNF-. Instead, GAPDH was used for RNA interference and TNF- experiments. 2-MG has been shown not to be affected by insulin and lipodystrophy (26,27) and was not affected by the other conditions used in the current study. The mRNA levels of the different genes were determined as previously described (25) and expressed as corrected threshold cycle (Ct) values or as ratios to 2-MG or GAPDH, as indicated. To clarify the data not expressed as percent of control, the ratio of CIDEA to 2-MG in obese humans (before weight reduction) and the mRNA levels in BAT from C57BL/6J mice on normal diet were set to 1, and the other values were expressed relative to these ratios or levels. The primer pairs for SYBR Green-based quantitative real-time PCR (Invitrogen, Tstrup, Denmark) were designed to span exon-intron boundaries and generate a single amplicon. Dissociation curves and agarose gel electrophoresis were used to check for a single product, and BLAST (basic local alignment search tool) searches were performed to ensure that the primer pairs were specific for the different genes. Thus, the CIDEA primers did not display any homology with other genes in the CIDE family, such as FSP27/CIDE3. The forward and reverse primers were for human CIDEA 5'-CATGTATGAGATGTACTCCGTGTC-3' and 5'-GAGTAGGACAGGAACCGCAG-3', for human MCP-1 5'-GTGTCCCAAAGAAGCTGTGA-3' and 5'-GTTTGCTTGTCCAGGTGGT-3', for human 2-MG 5'-TGCTGTCTCCATGTTTGATGTATCT-3' and 5'-TCTCTGCTCCCCACCTCTAAGT-3', for mouse CIDEA 5'-AAAGGGACAGAAATGGACAC-3' and 5'-TTGAGACAGCCGAGGAAG-3', and for mouse 2-MG 5'-CATGGCTCGCTCGGTGAC-3' and 5'-CAGTTCAGTATGTTCGGCTTCC-3'. The primer sequences for human GAPDH and human PLIN have been described (25). All samples were run in duplicate or triplicate, and standard curves created by repeated dilutions of cDNA were used to check the PCR efficiency and reproducibility.
Statistical analysis.
Data are the means ± SE. Statistical significance was determined by nonparametric methods because we could not ensure normal distribution of the parameters investigated. The Mann-Whitney U test, Wilcoxon’s signed-rank test, Kruskal-Wallis test, and Spearman’s correlation test were used. P < 0.05 was considered significant.
RESULTS
CIDEA is expressed in human WAT.
We measured CIDEA mRNA in different WAT preparations from nine obese healthy women (aged 26eC57 years, BMI 31eC46 kg/m2) and eight nonobese healthy subjects (five female, three male, aged 24eC42 years, BMI <30 kg/m2). The mRNA levels in adipose tissue correlated strongly with those in isolated adipocytes from the same subjects (P = 0.0008) (Fig. 1A). The ratio of CIDEA to 2-MG mRNA was 6 times higher in cells than in tissue (P = 0.0003), mainly because of higher 2-MG levels in tissue (1.2 ± 0.09 and 5.4 ± 0.4 in cells and tissue, respectively). When we compared the CIDEA values without correcting for -2 microglobulin, the correlation was even stronger (P = 0.0006, values not shown).
CIDEA mRNA expression correlates with features of the metabolic syndrome.
CIDEA mRNA was determined in subcutaneous WAT from 143 obese (BMI 38 ± 0.4 kg/m2) and 43 nonobese (BMI 24 ± 0.5 kg/m2) healthy subjects (Fig. 1B). The obese subjects were insulin resistant compared with the nonobese subjects (P < 0.0001, data not shown), as evidenced by increased values for the insulin sensitivity index HOMA (18). Furthermore, their basal lipolytic activity in subcutaneous WAT was increased twofold (P < 0.001, data not shown). CIDEA mRNA levels were 50% lower in the obese compared with the nonobese subjects (P = 0.0004), with an opposite change of GAPDH (P < 0.0001). We assessed the effect of weight reduction on CIDEA mRNA in 12 obese subjects (9 women, 3 men) undergoing bariatric surgery. At the follow-up 2eC4 years after surgery, they were weight stable, and their BMI had decreased by an average of 15 kg/m2 (Fig. 1C) (P = 0.002). At the same time, insulin sensitivity (HOMA) was improved (P < 0.01, data not shown), and the rate of basal adipocyte lipolysis in subcutaneous WAT was reduced by 40% (P < 0.05, values not shown). After weight reduction the CIDEA mRNA levels were almost doubled (Fig. 1D) (P = 0.02).
The relationship between clinical phenotype and CIDEA expression was investigated by dividing the 186 subjects described above into tertiles (n = 62) based on high (1.20 ± 0.15), intermediate (0.46 ± 0.01), or low (0.23 ± 0.01) ratios of CIDEA to 2-MG mRNA (Fig. 2AeCD). Waist circumference, fat cell volume, insulin resistance, and basal lipolytic activity in adipose tissue were highest in the lowest CIDEA tertile and lowest in the upper tertile. In general, intermediate values were recorded in the middle tertile (P 0.005).
CIDEA is downregulated by TNF- via JNK.
TNF- is associated with the metabolic syndrome because it stimulates lipolysis, is overexpressed in obesity, and involved in obesity-induced insulin resistance (24). We investigated whether TNF- stimulation affects CIDEA expression in fat cells. CIDEA mRNA levels in differentiated preadipocytes were markedly downregulated by a 48-h incubation in 100 ng/ml TNF- (Fig. 3A) (P = 0.012, n = 8). This decrease was paralleled by a two- to threefold increase in basal lipolysis in the same cell preparations (11).
The MAPKs p38, p44/42, and JNK are activated by TNF- in human fat cells (11,24,28). To further investigate the signaling pathway(s) between CIDEA and TNF-, we therefore incubated differentiated preadipocytes with TNF- in the absence or presence of specific MAPK inhibitors. The inhibitors of p44/42 (PD98059) and p38 (SB203580) did not affect the TNF--induced downregulation of CIDEA mRNA (data not shown), but the JNK inhibitor (SP600125) completely counteracted the downregulation of CIDEA in a concentration-dependent fashion (Fig. 3B). A maximum effect occurred at 6.7 eol/l.
Effect of CIDEA depletion on basal lipolysis, TNF- secretion, and other TNF- targets.
Because TNF- downregulates CIDEA expression and at the same time stimulates basal lipolysis, we investigated the putative effects of CIDEA by gene silencing. The quantitative relationship between lipolysis and CIDEA expression was determined in vitro by treating preadipocytes with siRNA duplexes directed against human CIDEA and measuring basal lipolysis as glycerol release into the medium. Using preadipocytes from nine healthy women (aged 47.0 ± 4.6 years, range 30eC70; BMI 26.5 ± 0.8 kg/m2, range 22.9eC30.2), we performed 24-h incubations with different amounts of siRNA, which resulted in a 20eC95% decrease in CIDEA expression. No effect on CIDEA mRNA levels was observed when cells were treated with siRNA without known similarities to human sequences or when cells were treated with transfection reagent only. Basal lipolysis was measured in six of these experiments. The changes in CIDEA mRNA and glycerol release showed a strong inverse correlation (Fig. 3C) (P = 0.013), indicating that CIDEA downregulation stimulates lipolysis.
To assess whether CIDEA depletion influenced TNF- action, the levels of secreted TNF- protein in cell media from eight of the RNA interference experiments were measured. There was a strong inverse correlation between the levels of secreted TNF- protein and the ratio of CIDEA to GAPDH mRNA (Fig. 3D) (P = 0.0051). The secreted levels of TNF- also correlated with the glycerol release from the same cells (P = 0.03) (graph not shown). In the samples where CIDEA mRNA was inhibited to <10% of control (n = 2), TNF- secretion was increased as much as fourfold.
To determine whether the effect of CIDEA silencing on TNF- secretion was transcriptional or posttranscriptional, we measured the TNF- mRNA levels in the siRNA-treated cells (n = 8). There was no correlation between the ratio of TNF- to GAPDH mRNA and the ratio of CIDEA to GAPDH mRNA (Fig. 4A) (P = 0.29). To assess whether CIDEA downregulation resulted in a selective effect on lipolysis by TNF-, additional measurements were performed in the siRNA-treated cells and incubation media. Adipocytes secrete a number of proteins in addition to TNF- (rev. in 29), among them adiponectin, which is inhibited by TNF- (30). We determined the amount of adiponectin secreted into the media of the cells in the same experiments as in Fig. 3D. The CIDEA mRNA levels in these experiments did not correlate with the amount of adiponectin (P = 0.2, graph not shown).
Because the lipid droplet-associated phosphoprotein perilipin plays an important role in basal lipolysis (31,32), PLIN mRNA levels were assessed in the siRNA-treated cells with the most marked CIDEA downregulation (by 75eC95%, n = 4). In these samples PLIN mRNA levels were 50% lower than in control cells (Fig. 4B) (P = 0.03). The perilipin protein levels were determined by Western blot in four RNA interference experiments. In contrast to mRNA data, there was no significant downregulation of the protein (values not shown).
MCP-1 is upregulated by TNF- in human preadipocytes (33) and 3T3-L1 cells (34). We therefore measured the MCP-1 mRNA levels and secretion of MCP-1 protein in the same RNA interference experiments as in Fig. 3C. The ratio of MCP-1 to GAPDH mRNA did not correlate with the ratio of CIDEA to GAPDH mRNA (Fig. 4C) (P = 0.53). Likewise, the amount of MCP-1 protein secreted into the cell media did not correlate with the ratio of CIDEA to GAPDH mRNA levels (Fig. 4D) (P = 0.38).
CIDEA expression in mice differs qualitatively from that in humans.
To study the species differences in CIDEA expression, we measured the mRNA levels of CIDEA in WAT and BAT from 18 mice of three different strains (C57BL/6J, 129Sv/Pas, and NMRI) that were divided into two groups and fed either normal or cafeteria diet. Data from the C57BL/6J mice on normal diet are shown in Fig. 5AeCB and are representative of all three strains. CIDEA mRNA in WAT was not detectable with SYBR Green-based quantitative real-time PCR but highly so in BAT, confirming earlier studies (16) (Fig. 5A). The mRNA expression of 2-MG in the same samples showed the opposite pattern, with much higher levels in WAT than in BAT (Fig. 5B). Using the more sensitive TaqMan probes, we found 400 times less CIDEA mRNA in WAT as compared with BAT (data not shown). This low level of expression could be nonspecific for fat cells and was not further analyzed. There was a significant diet effect on body weight increase in C57BL/6J mice (P < 0.05) and a borderline significant increase in NMRI mice (Fig. 5C) (P = 0.06). Surprisingly, the body weight increase in 129Sv/Pas mice on normal diet was marginally larger than for the mice on cafeteria diet (Fig. 5C), even though food consumption was equally increased in all three strains (values not shown). There was no effect of the diet or body weight increase in any strain regarding CIDEA mRNA (Fig. 5D).
DISCUSSION
The current study demonstrates that CIDEA is expressed in human white fat cells and that the adipose mRNA levels of CIDEA are markedly influenced by body fat status. This is in clear contrast to mice, where the gene is expressed in brown fat cells only and not influenced by obesity. In human adipose tissue, the expression of CIDEA is downregulated by obesity and normalized after weight reduction. The mRNA levels of CIDEA are inversely correlated to perturbations that are characteristic for the metabolic syndrome: abdominal obesity, enlarged fat cells, insulin resistance (high HOMA index), and increased basal lipolysis (glycerol release). The metabolic syndrome also has other clinical features not measured here, but it is nevertheless obvious that CIDEA mRNA in human WAT is strongly associated with the metabolic syndrome.
Although the RNA interference technique (rev. in 35) has been successfully used in 3T3-L1 preadipocytes (36,37), its usefulness in human preadipocytes has not been reported before. In our experiments, CIDEA mRNA knockdown varied between 20 and 95%, probably because of the interindividual differences of the primary cells used. The effect of CIDEA RNA interference on glycerol release from the preadipocytes correlated with the degree of mRNA downregulation. Our experiments suggest that basal lipolysis in human preadipocytes is under tonic inhibition by CIDEA and that relief of inhibition by mRNA depletion augments lipolysis. This effect most probably involves posttranscriptional modification of TNF- because the release of this prolipolytic cytokine is stimulated by CIDEA downregulation, but there is no corresponding change in the TNF- mRNA levels. CIDEA is apparently a very strong inhibitor of TNF- secretion because adipocytes with >90% CIDEA depletion displayed a fourfold stimulated TNF- release. A similar downregulation of CIDEA also caused a marked decrease in PLIN mRNA but no detectable decrease in the protein amounts of perilipin. This, however, does not exclude perilipin as an indirect target for CIDEA, putatively through TNF-. It may, for example, necessitate a longer time to reduce the amount of protein than mRNA. Furthermore, we (25) and others (38) have previously shown discrepancies between perilipin mRNA and protein levels in human adipose tissue. In addition, CIDEA-TNF- interactions might involve translocation and/or phosphorylation of perilipin, which were not examined here.
CIDEA-TNF- interactions appear to be specific for lipolysis because the mRNA expression and protein secretion of a known target for TNF-, MCP-1, were not influenced by RNA interference. Also, the secretion of the adipose-specific protein adiponectin, whose expression has been shown to be regulated by (39) or associated with (40) TNF- levels, was not affected. At present we do not know why only glycerol release and not secretion of adiponectin or MCP-1 was influenced by increased TNF- release after CIDEA depletion. However, it is possible that glycerol stimulation is an early event, and effects of increased TNF- levels on adiponectin and MCP-1 secretion occur at time points later than 24 h.
The signaling pathways of TNF- to lipolysis are very complex and involve signaling through MAPKs, resulting in downregulation of Gi (inhibitory G-protein -subunit) as well as changes in perilipin phosphorylation and expression (11,24,28,41). In the current study, we have shown that TNF- inhibits CIDEA expression and that this downregulation of CIDEA mRNA is mediated by JNK, one of the MAPKs that is involved in TNF--induced stimulation of lipolysis in human fat cells (24). It is noteworthy that the concentration-dependent relationship for the effect of the JNK inhibitor SP600125 on CIDEA was almost identical to that previously shown on lipolysis using an experimental setting similar to the current study (11). The other two MAPKs that are also active in human fat cells, p44/42 and p38 (11,24,28), do not seem to be involved in regulating CIDEA expression. Our data suggest a reciprocal inhibition of CIDEA and TNF- because CIDEA depletion by siRNA causes an increased secretion of TNF- protein. It is tempting to speculate that the ability of TNF- to inhibit CIDEA is an indirect way for this cytokine to further stimulate lipolysis by counteracting the inhibitory effects of CIDEA on TNF- release. Conversely, the direct way would be through signaling to perilipin and/or other lipolytic regulators.
In the current study, we have only investigated effects of CIDEA depletion. Studies of stable overexpression would require human fat cell lines that are not available. A human fat cell line derived from BAT (42) differs in many qualitative parameters from white adipocytes and can therefore not be used. Transient overexpression is not useful because such ectopic CIDEA expression causes excessive apoptosis in other cell systems (12).
In accordance with previous studies (16), we found that CIDEA is virtually not expressed in WAT in mice. Therefore, CIDEA expression is completely different in mice compared with humans. Humans have easily detectable levels in both WAT and isolated adipocytes. An interesting observation is that the mouse strains displaying low CIDEA levels (C57BL/6J and NMRI) were those that developed diet-induced obesity. It has been suggested that CIDEA may induce obesity through processes in BAT of mice (43), but our results would rather indicate a reverse correlation.
Based on the current results, we propose the following mode of action of CIDEA in human WAT (Fig. 6): CIDEA cross-talks with TNF-, and this has consequences for lipolysis. CIDEA decreases the availability of TNF- by inhibiting cytokine secretion predominately through posttranscriptional mechanisms, which in turn counteracts the ability of TNF- to stimulate lipolysis. TNF- downregulates the expression of CIDEA through signaling via JNK, which in turn increases the availability of TNF- and thereby lipolytic stimulation. This cross-talk seems to be specific for humans. In obesity CIDEA expression is downregulated probably because of increased TNF- action. This elevates the basal lipolytic activity and could be an important contributing factor to elevated circulating fatty acid levels in obesity. Other adipocyte factors responsible for increased basal lipolysis could be additional cytokines and insulin resistance in the fat cells. Because CIDEA is expressed in adipocytes of WAT, it is possible that cell-specific pharmacological agents that either upregulate CIDEA expression or alter the interactions between TNF- and CIDEA (such as JNK inhibitors) could be useful therapeutic agents to decrease fatty acid levels in obese individuals and thereby ameliorate the disadvantageous effects of these lipids.
ACKNOWLEDGMENTS
This study was supported by grants from the Swedish Research Council, the Swedish Diabetes Association, the Swedish Heart and Lung Foundation, the King Gustaf V and Queen Victoria Foundation, the Diabetes Research and Wellness Foundation, the Swedish Cancer Foundation, and EFSD (European Foundation for the Study of Diabetes)/Novo Nordisk Diabetes Research.
FOOTNOTES
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
BAT, brown adipose tissue; CIDEA, cell death-inducing DFFA (DNA fragmentation factor-)-like effector A; HOMA, homeostasis model assessment; JNK, c-Jun NH2-terminal kinase; MAPK, mitogen-activated protein kinase; MCP-1, monocyte chemoattractant protein-1; siRNA, short interfering RNA; TNF-, tumor necrosis factor-; WAT, white adipose tissue
REFERENCES
Hill JO, Melanson EL, Wyatt HT: Dietary fat intake and regulation of energy balance: implications for obesity. J Nutr 130:284SeC288S, 2000
Caterson ID, Gill TP: Obesity: epidemiology and possible prevention. Best Pract Res Clin Endocrinol Metab 16:595eC610, 2002
Stein CJ, Colditz GA: The epidemic of obesity. J Clin Endocrinol Metab 89:2522eC2525, 2004
Carr MC, Brunzell JD: Abdominal obesity and dyslipidemia in the metabolic syndrome: importance of type 2 diabetes and familial combined hyperlipidemia in coronary artery disease risk. J Clin Endocrinol Metab 89:2601eC2607, 2004
Large V, Arner P: Regulation of lipolysis in humans. Pathophysiological modulation in obesity, diabetes, and hyperlipidaemia. Diabetes Metab 24:409eC418, 1998
Blaak EE: Fatty acid metabolism in obesity and type 2 diabetes mellitus. Proc Nutr Soc 62:753eC760, 2003
Hotamisligil GS, Arner P, Caro JF, Atkinson RL, Spiegelman BM: Increased adipose tissue expression of tumor necrosis factor-alpha in human obesity and insulin resistance. J Clin Invest 95:2409eC2415, 1995
Green A, Dobias SB, Walters DJ, Brasier AR: Tumor necrosis factor increases the rate of lipolysis in primary cultures of adipocytes without altering levels of hormone-sensitive lipase. Endocrinology 134:2581eC2588, 1994
Souza SC, Yamamoto MT, Franciosa MD, Lien P, Greenberg AS: BRL 49653 blocks the lipolytic actions of tumor necrosis factor-alpha: a potential new insulin-sensitizing mechanism for thiazolidinediones. Diabetes 47:691eC695, 1998
Hauner H, Petruschke T, Russ M, Rohrig K, Eckel J: Effects of tumour necrosis factor alpha (TNF alpha) on glucose transport and lipid metabolism of newly-differentiated human fat cells in cell culture. Diabetologia 38:764eC771, 1995
Ryden M, Arvidsson E, Blomqvist L, Perbeck L, Dicker A, Arner P: Targets for TNF-alpha-induced lipolysis in human adipocytes. Biochem Biophys Res Commun 318:168eC175, 2004
Inohara N, Koseki T, Chen S, Wu X, Nunez G: CIDE, a novel family of cell death activators with homology to the 45 kDa subunit of the DNA fragmentation factor. EMBO J 17:2526eC2533, 1998
Reed JC, Doctor K, Rojas A, Zapata JM, Stehlik C, Fiorentino L, Damiano J, Roth W, Matsuzawa S, Newman R, Takayama S, Marusawa H, Xu F, Salvesen G, Godzik A: Comparative analysis of apoptosis and inflammation genes of mice and humans. Genome Res 13:1376eC1388, 2003
Danesch U, Hoeck W, Ringold GM: Cloning and transcriptional regulation of a novel adipocyte-specific gene, FSP27: CAAT-enhancer-binding protein (C/EBP) and C/EBP-like proteins interact with sequences required for differentiation-dependent expression. J Biol Chem 267:7185eC7193, 1992
Williams PM, Chang DJ, Danesch U, Ringold GM, Heller RA: CCAAT/enhancer binding protein expression is rapidly extinguished in TA1 adipocyte cells treated with tumor necrosis factor. Mol Endocrinol 6:1135eC1141, 1992
Zhou Z, Yon Toh S, Chen Z, Guo K, Ng CP, Ponniah S, Lin SC, Hong W, Li P: Cidea-deficient mice have lean phenotype and are resistant to obesity. Nat Genet 35:49eC56, 2003
Lofgren P, Hoffstedt J, Ryden M, Thorne A, Holm C, Wahrenberg H, Arner P: Major gender differences in the lipolytic capacity of abdominal subcutaneous fat cells in obesity observed before and after long-term weight reduction. J Clin Endocrinol Metab 87:764eC771, 2002
Emoto M, Nishizawa Y, Maekawa K, Hiura Y, Kanda H, Kawagishi T, Shoji T, Okuno Y, Morii H: Homeostasis model assessment as a clinical index of insulin resistance in type 2 diabetic patients treated with sulfonylureas. Diabetes Care 22:818eC822, 1999
Hoffstedt J, Arvidsson E, Sjolin E, Wahlen K, Arner P: Adipose tissue adiponectin production and adiponectin serum concentration in human obesity and insulin resistance. J Clin Endocrinol Metab 89:1391eC1396, 2004
Hellmer J, Marcus C, Sonnenfeld T, Arner P: Mechanisms for differences in lipolysis between human subcutaneous and omental fat cells. J Clin Endocrinol Metab 75:15eC20, 1992
Arner P: Relationship between intracellular cyclic AMP and lipolysis in human adipose tissue. Acta Med Scand 200:179eC186, 1976
Reynisdottir S, Wahrenberg H, Carlstrom K, Rossner S, Arner P: Catecholamine resistance in fat cells of women with upper-body obesity due to decreased expression of beta 2-adrenoceptors. Diabetologia 37:428eC435, 1994
van Harmelen V, Dicker A, Ryden M, Hauner H, Lonnqvist F, Naslund E, Arner P: Increased lipolysis and decreased leptin production by human omental as compared with subcutaneous preadipocytes. Diabetes 51:2029eC2036, 2002
Ryden M, Dicker A, van Harmelen V, Hauner H, Brunnberg M, Perbeck L, Lonnqvist F, Arner P: Mapping of early signaling events in tumor necrosis factor-alpha-mediated lipolysis in human fat cells. J Biol Chem 277:1085eC1091, 2002
Arvidsson E, Blomqvist L, Ryden M: Depot-specific differences in perilipin mRNA but not protein expression in obesity. J Intern Med 255:595eC601, 2004
Kannisto K, Sutinen J, Korsheninnikova E, Fisher RM, Ehrenborg E, Gertow K, Virkamaki A, Nyman T, Vidal H, Hamsten A, Yki-Jarvinen H: Expression of adipogenic transcription factors, peroxisome proliferator-activated receptor gamma co-activator 1, IL-6 and CD45 in subcutaneous adipose tissue in lipodystrophy associated with highly active antiretroviral therapy. AIDS 17:1753eC1762, 2003
Lin YJ, Harada S, Loten EG, Smith RM, Jarett L: Direct stimulation of immediate-early genes by intranuclear insulin in trypsin-treated H35 hepatoma cells. Proc Natl Acad Sci U S A 89:9691eC9694, 1992
Zhang HH, Halbleib M, Ahmad F, Manganiello VC, Greenberg AS: Tumor necrosis factor-alpha stimulates lipolysis in differentiated human adipocytes through activation of extracellular signal-related kinase and elevation of intracellular cAMP. Diabetes 51:2929eC2935, 2002
Wiecek A, Kokot F, Chudek J, Adamczak M: The adipose tissue—a novel endocrine organ of interest to the nephrologist. Nephrol Dial Transplant 17:191eC195, 2002
Maeda N, Takahashi M, Funahashi T, Kihara S, Nishizawa H, Kishida K, Nagaretani H, Matsuda M, Komuro R, Ouchi N, Kuriyama H, Hotta K, Nakamura T, Shimomura I, Matsuzawa Y: PPAR ligands increase expression and plasma concentrations of adiponectin, an adipose-derived protein. Diabetes 50:2094eC2099, 2001
Souza SC, Muliro KV, Liscum L, Lien P, Yamamoto MT, Schaffer JE, Dallal GE, Wang X, Kraemer FB, Obin M, Greenberg AS: Modulation of hormone-sensitive lipase and protein kinase A-mediated lipolysis by perilipin A in an adenoviral reconstituted system. J Biol Chem 277:8267eC8272, 2002
Tansey JT, Sztalryd C, Gruia-Gray J, Roush DL, Zee JV, Gavrilova O, Reitman ML, Deng CX, Li C, Kimmel AR, Londos C: Perilipin ablation results in a lean mouse with aberrant adipocyte lipolysis, enhanced leptin production, and resistance to diet-induced obesity. Proc Natl Acad Sci U S A 98:6494eC6499, 2001
Wang B, Jenkins JR, Trayhurn P: Expression and secretion of inflammation-related adipokines by human adipocytes differentiated in culture: integrated response to TNF-alpha. Am J Physiol Endocrinol Metab 288:E731eCE740, 2005
Sartipy P, Loskutoff DJ: Monocyte chemoattractant protein 1 in obesity and insulin resistance. Proc Natl Acad Sci U S A 100:7265eC7270, 2003
Hannon GJ, Rossi JJ: Unlocking the potential of the human genome with RNA interference. Nature 431:371eC378, 2004
Xu J, Liao K: Protein kinase B/AKT 1 plays a pivotal role in insulin-like growth factor-1 receptor signaling induced 3T3eCL1 adipocyte differentiation. J Biol Chem 279:35914eC35922, 2004
Mitra P, Zheng X, Czech MP: RNAi-based analysis of CAP, Cbl, and CrkII function in the regulation of GLUT4 by insulin. J Biol Chem 279:37431eC37435, 2004
Wang Y, Sullivan S, Trujillo M, Lee MJ, Schneider SH, Brolin RE, Kang YH, Werber Y, Greenberg AS, Fried SK: Perilipin expression in human adipose tissues: effects of severe obesity, gender, and depot. Obes Res 11:930eC936, 2003
Bruun JM, Lihn AS, Verdich C, Pedersen SB, Toubro S, Astrup A, Richelsen B: Regulation of adiponectin by adipose tissue-derived cytokines: in vivo and in vitro investigations in humans. Am J Physiol Endocrinol Metab 285:E527eCE533, 2003
Kern PA, Di Gregorio GB, Lu T, Rassouli N, Ranganathan G: Adiponectin expression from human adipose tissue: relation to obesity, insulin resistance, and tumor necrosis factor-alpha expression. Diabetes 52:1779eC1785, 2003
Gasic S, Tian B, Green A: Tumor necrosis factor alpha stimulates lipolysis in adipocytes by decreasing Gi protein concentrations. J Biol Chem 274:6770eC6775, 1999
Zilberfarb V, Pietri-Rouxel F, Jockers R, Krief S, Delouis C, Issad T, Strosberg AD: Human immortalized brown adipocytes express functional beta3-adrenoceptor coupled to lipolysis. J Cell Sci 110:801eC807, 1997
Lin SC, Li P: CIDE-A, a novel link between BAT and obesity. Trends Mol Med 10:434eC439, 2004(Elisabet Arvidsson Nordst)
2 Wenner-Gren Institute, Stockholm University, Stockholm, Sweden
3 Department of Surgery, Karolinska Institutet, Karolinska University Hospital Huddinge, Stockholm, Sweden
ABSTRACT
Elevated circulating fatty acid concentration is a hallmark of insulin resistance and is at least in part attributed to the action of adipose tissue-derived tumor necrosis factor- (TNF-) on lipolysis. Cell death-inducing DFFA (DNA fragmentation factor-)-like effector A (CIDEA) belongs to a family of proapoptotic proteins that has five known members in humans and mice. The action of CIDEA is unknown, but CIDEA-null mice are resistant to obesity and diabetes. We investigated CIDEA in adipose tissue of obese and lean humans and mice. The mRNA was expressed in white human fat cells and in brown mouse adipocytes. The adipose mRNA expression of CIDEA in mice was not influenced by obesity. However, CIDEA expression was decreased twofold in obese humans and normalized after weight reduction. Low adipose CIDEA expression was associated with several features of the metabolic syndrome. Human adipocyte depletion of CIDEA by RNA interference stimulated lipolysis and increased TNF- secretion by a posttranscriptional effect. Conversely, TNF- treatment decreased adipocyte CIDEA expression via the mitogen-activated protein kinase c-Jun NH2-terminal kinase. We propose an important and human-specific role for CIDEA in lipolysis regulation and metabolic complications of obesity, which is at least in part mediated by cross-talk between CIDEA and TNF-.
Obesity is an excess of body fat that develops from a combination of genetic and environmental factors, leading to a disturbed balance between energy intake and energy expenditure (1). The prevalence is increasing in both developed and developing countries and has reached epidemic proportions (2,3). Obesity, in particular abdominal obesity, is closely associated with insulin resistance, non-insulin-dependent diabetes (type 2 diabetes), dyslipidemia, and cardiovascular disease (4).
Elevated circulating fatty acid levels in obesity resulting from increased basal adipocyte lipolysis could cause perturbations such as insulin resistance and type 2 diabetes (5). The exact molecular mechanisms causing activation of basal lipolysis are so far unknown (6), although the cytokine tumor necrosis factor- (TNF-) is a potential candidate. The production of adipose tissue-derived TNF- is increased in human obesity (7), and TNF- stimulates adipocyte lipolysis in rodent (8,9) and human fat cells (10,11).
The mammalian gene cell death-inducing DFFA (DNA fragmentation factor-)-like effector A (CIDEA) was recently identified and characterized (12). CIDEA belongs to a family of proapoptotic proteins (12) that has five known members in the transcriptomes of humans and mice (13). CIDEA is highly homologous to FSP27 (12), an adipose-specific gene whose expression is associated with terminal differentiation of fat cells (14). The expression of FSP27 (also called clone 47) is regulated by the tumor necrosis pathway (15).
Although the murine expression pattern of CIDEA has been studied and CIDEA-null mice are resistant to diet-induced obesity and diabetes (16), little else is known regarding the function of the CIDEA gene. CIDEA transcripts of various lengths are present in different human tissues (e.g., heart, skeletal muscle, brain, placenta, and kidney) (12), but there are no reports about CIDEA in human adipose tissue. Preliminary results from microarray studies in our laboratory demonstrated that CIDEA is significantly expressed in human white adipose tissue (WAT). This is in contrast to mice, where CIDEA is expressed in brown adipose tissue (BAT) but not detectable in WAT (16). We investigated the role of this gene in human WAT and obesity and show a specific role for CIDEA in regulating lipolysis in white human fat cells. We found interactions between CIDEA and TNF- that may be linked to the insulin-resistant phenotype of many obese subjects.
RESEARCH DESIGN AND METHODS
One cohort consisted of 40 men and 146 women, all healthy and free of medication but with a large interindividual range in BMI (18eC50 kg/m2). The subjects were divided into an obese (n = 143, BMI >30 kg/m2) and a nonobese (n = 43) subgroup. Twelve of the obese healthy subjects (BMI 35eC50 kg/m2) underwent gastric banding to treat their obesity. Fat tissue samples were obtained before and 2eC4 years after surgery in a weight-stable condition. These subjects were part of a larger cohort described previously (17). All subjects were examined at 8 A.M. after an overnight fast. Their height, weight, and waist circumference were measured. Venous plasma samples were drawn for the analysis of insulin and glucose and calculation of the homeostasis model assessment (HOMA) index ([plasma glucose (mmol/l) x plasma insulin (mU/l)]/22.5) (18), an indirect measure of in vivo insulin sensitivity. Thereafter, needle biopsies of subcutaneous adipose tissue (1eC2 g) were obtained under local anesthesia. For experimental studies, subcutaneous adipose tissue was obtained from healthy subjects after elective cosmetic liposuction. The studies were approved by the ethical committee at Karolinska Institutet and explained in detail to each of the subjects, from whom informed consent was obtained.
Mice.
A total of 18 male 7-week-old mice of three different strains (NMRI, C57BL/6J, and 129Sv/Pas, n = 6 from each strain; Scanbur B&K Universal, Sollentuna, Sweden) were fed normal pelleted chow (R70 Standard Diet; Lactamin, Vadstena, Sweden) with or without the addition of cafeteria diet ad libitum for 15 weeks. The mice had free access to water and were kept on a 12/12 h light/dark cycle. They were then killed by carbon dioxide anesthesia followed by cervical dislocation. The epididymal fat pads and interscapular BAT were dissected and snap frozen in liquid nitrogen for subsequent RNA isolation. All animal experimentation was performed in accordance with institutional guidelines, and ethical permission was obtained from the Northern Stockholm animal ethics committee.
Human adipose tissue and preadipocyte culture.
Adipose tissue (1 g) was immediately frozen in liquid nitrogen for subsequent RNA analysis. Fresh adipose tissue pieces were incubated as described for 2 h (19). Glycerol release to the medium was determined and related to the number of incubated fat cells, as previously described (20). Glycerol release is linear with incubation time for at least 4 h (21). Tissue was collagenase treated and mean fat cell size and mean fat cell weight determined as previously described (22). Preadipocytes were isolated from the stromal fraction of adipose tissue and differentiated into adipocytes as previously described (23), reaching full differentiation after 12eC14 days. The cells were seeded out in 12-well plates at a density of 30,000 cells/cm2. For studies of TNF--induced CIDEA downregulation, preadipocytes were incubated in the presence of 100 ng/ml TNF- in combination with different concentrations of the specific mitogen-activated protein kinase (MAPK) inhibitors SP600125 (for c-Jun NH2-terminal kinase [JNK]), PD98059 (for p44/42, also termed extracellular signal-regulated kinase 1/2), and SB203580 (for stress/cytokine-activated kinase, or p38) (all from Sigma, St Louis, MO) as described before (11,24). The MAPK cascades activate different transcription factors that mediate TNF--regulated gene expression (24). TNF- was added on day 10 (1 h after addition of the inhibitors) or on day 12 (alone), and the cells were incubated for 48 h before RNA isolation. Control cells were incubated in medium alone. Lipolysis in preadipocytes was assessed as glycerol release into the medium, as previously described (20).
RNA interference and protein analysis.
Optimal transfection conditions of human preadipocytes were initially determined by titrating different amounts of fluorescent short interfering RNA (siRNA) duplexes and the transfection reagent RNAiFect (both from Qiagen, Hilden, Germany). Analysis by fluorescence microscopy 4eC12 h later showed a transfection efficiency ranging from 5 to 70%, depending on the different conditions. These experiments served as a basis for optimizing CIDEA gene silencing. Cells at day 8 of differentiation (when the cells have developed lipid droplets but are not fully differentiated) were transfected with or without 1 or 2 e of CIDEA siRNA (Qiagen, Hilden, Germany) and incubated for 24 h, a time point where a significant gene silencing effect was observed. Cells incubated without siRNA served as controls. To control for nonspecific gene silencing effects, parallel cells were transfected with siRNA without known similarities to human sequences or with CIDEA siRNA without RNAiFect. Conditioned cell media aliquots were then analyzed for glycerol content while cells were lysed for RNA or protein isolation. Perilipin protein levels were detected by Western blot and related to -actin protein levels, as previously described (11). Protein release into the cell media was determined by a radioimmunoassay for adiponectin (Linco, St Charles, MO) and with Quantikine human immunoassays (R&D Systems, Abingdon, U.K.) for TNF- and monocyte chemoattractant protein-1 (MCP-1). The kits were used according to instructions from the manufacturers.
mRNA quantitation.
Total RNA was extracted from human or mouse adipose tissue, differentiated preadipocytes, or isolated adipocytes and reverse-transcribed as previously described (25). Quantitative real-time PCR was performed in an iCycler IQ (Bio-Rad Laboratories, Hercules, CA), using the less expensive SYBR Green-based technology for CIDEA quantitation in large cohorts (human and mouse) and mRNA levels of perilipin (PLIN), MCP-1, and the reference genes -2 microglobulin (2-MG; human and mouse) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). TaqMan probes (Applied Biosystems, Foster City, CA) were used for CIDEA and TNF- measurements in smaller experiments. 2-MG was used as a reference gene except for the TNF--treated samples, where 2-MG in our hands appears to be regulated by TNF-. Instead, GAPDH was used for RNA interference and TNF- experiments. 2-MG has been shown not to be affected by insulin and lipodystrophy (26,27) and was not affected by the other conditions used in the current study. The mRNA levels of the different genes were determined as previously described (25) and expressed as corrected threshold cycle (Ct) values or as ratios to 2-MG or GAPDH, as indicated. To clarify the data not expressed as percent of control, the ratio of CIDEA to 2-MG in obese humans (before weight reduction) and the mRNA levels in BAT from C57BL/6J mice on normal diet were set to 1, and the other values were expressed relative to these ratios or levels. The primer pairs for SYBR Green-based quantitative real-time PCR (Invitrogen, Tstrup, Denmark) were designed to span exon-intron boundaries and generate a single amplicon. Dissociation curves and agarose gel electrophoresis were used to check for a single product, and BLAST (basic local alignment search tool) searches were performed to ensure that the primer pairs were specific for the different genes. Thus, the CIDEA primers did not display any homology with other genes in the CIDE family, such as FSP27/CIDE3. The forward and reverse primers were for human CIDEA 5'-CATGTATGAGATGTACTCCGTGTC-3' and 5'-GAGTAGGACAGGAACCGCAG-3', for human MCP-1 5'-GTGTCCCAAAGAAGCTGTGA-3' and 5'-GTTTGCTTGTCCAGGTGGT-3', for human 2-MG 5'-TGCTGTCTCCATGTTTGATGTATCT-3' and 5'-TCTCTGCTCCCCACCTCTAAGT-3', for mouse CIDEA 5'-AAAGGGACAGAAATGGACAC-3' and 5'-TTGAGACAGCCGAGGAAG-3', and for mouse 2-MG 5'-CATGGCTCGCTCGGTGAC-3' and 5'-CAGTTCAGTATGTTCGGCTTCC-3'. The primer sequences for human GAPDH and human PLIN have been described (25). All samples were run in duplicate or triplicate, and standard curves created by repeated dilutions of cDNA were used to check the PCR efficiency and reproducibility.
Statistical analysis.
Data are the means ± SE. Statistical significance was determined by nonparametric methods because we could not ensure normal distribution of the parameters investigated. The Mann-Whitney U test, Wilcoxon’s signed-rank test, Kruskal-Wallis test, and Spearman’s correlation test were used. P < 0.05 was considered significant.
RESULTS
CIDEA is expressed in human WAT.
We measured CIDEA mRNA in different WAT preparations from nine obese healthy women (aged 26eC57 years, BMI 31eC46 kg/m2) and eight nonobese healthy subjects (five female, three male, aged 24eC42 years, BMI <30 kg/m2). The mRNA levels in adipose tissue correlated strongly with those in isolated adipocytes from the same subjects (P = 0.0008) (Fig. 1A). The ratio of CIDEA to 2-MG mRNA was 6 times higher in cells than in tissue (P = 0.0003), mainly because of higher 2-MG levels in tissue (1.2 ± 0.09 and 5.4 ± 0.4 in cells and tissue, respectively). When we compared the CIDEA values without correcting for -2 microglobulin, the correlation was even stronger (P = 0.0006, values not shown).
CIDEA mRNA expression correlates with features of the metabolic syndrome.
CIDEA mRNA was determined in subcutaneous WAT from 143 obese (BMI 38 ± 0.4 kg/m2) and 43 nonobese (BMI 24 ± 0.5 kg/m2) healthy subjects (Fig. 1B). The obese subjects were insulin resistant compared with the nonobese subjects (P < 0.0001, data not shown), as evidenced by increased values for the insulin sensitivity index HOMA (18). Furthermore, their basal lipolytic activity in subcutaneous WAT was increased twofold (P < 0.001, data not shown). CIDEA mRNA levels were 50% lower in the obese compared with the nonobese subjects (P = 0.0004), with an opposite change of GAPDH (P < 0.0001). We assessed the effect of weight reduction on CIDEA mRNA in 12 obese subjects (9 women, 3 men) undergoing bariatric surgery. At the follow-up 2eC4 years after surgery, they were weight stable, and their BMI had decreased by an average of 15 kg/m2 (Fig. 1C) (P = 0.002). At the same time, insulin sensitivity (HOMA) was improved (P < 0.01, data not shown), and the rate of basal adipocyte lipolysis in subcutaneous WAT was reduced by 40% (P < 0.05, values not shown). After weight reduction the CIDEA mRNA levels were almost doubled (Fig. 1D) (P = 0.02).
The relationship between clinical phenotype and CIDEA expression was investigated by dividing the 186 subjects described above into tertiles (n = 62) based on high (1.20 ± 0.15), intermediate (0.46 ± 0.01), or low (0.23 ± 0.01) ratios of CIDEA to 2-MG mRNA (Fig. 2AeCD). Waist circumference, fat cell volume, insulin resistance, and basal lipolytic activity in adipose tissue were highest in the lowest CIDEA tertile and lowest in the upper tertile. In general, intermediate values were recorded in the middle tertile (P 0.005).
CIDEA is downregulated by TNF- via JNK.
TNF- is associated with the metabolic syndrome because it stimulates lipolysis, is overexpressed in obesity, and involved in obesity-induced insulin resistance (24). We investigated whether TNF- stimulation affects CIDEA expression in fat cells. CIDEA mRNA levels in differentiated preadipocytes were markedly downregulated by a 48-h incubation in 100 ng/ml TNF- (Fig. 3A) (P = 0.012, n = 8). This decrease was paralleled by a two- to threefold increase in basal lipolysis in the same cell preparations (11).
The MAPKs p38, p44/42, and JNK are activated by TNF- in human fat cells (11,24,28). To further investigate the signaling pathway(s) between CIDEA and TNF-, we therefore incubated differentiated preadipocytes with TNF- in the absence or presence of specific MAPK inhibitors. The inhibitors of p44/42 (PD98059) and p38 (SB203580) did not affect the TNF--induced downregulation of CIDEA mRNA (data not shown), but the JNK inhibitor (SP600125) completely counteracted the downregulation of CIDEA in a concentration-dependent fashion (Fig. 3B). A maximum effect occurred at 6.7 eol/l.
Effect of CIDEA depletion on basal lipolysis, TNF- secretion, and other TNF- targets.
Because TNF- downregulates CIDEA expression and at the same time stimulates basal lipolysis, we investigated the putative effects of CIDEA by gene silencing. The quantitative relationship between lipolysis and CIDEA expression was determined in vitro by treating preadipocytes with siRNA duplexes directed against human CIDEA and measuring basal lipolysis as glycerol release into the medium. Using preadipocytes from nine healthy women (aged 47.0 ± 4.6 years, range 30eC70; BMI 26.5 ± 0.8 kg/m2, range 22.9eC30.2), we performed 24-h incubations with different amounts of siRNA, which resulted in a 20eC95% decrease in CIDEA expression. No effect on CIDEA mRNA levels was observed when cells were treated with siRNA without known similarities to human sequences or when cells were treated with transfection reagent only. Basal lipolysis was measured in six of these experiments. The changes in CIDEA mRNA and glycerol release showed a strong inverse correlation (Fig. 3C) (P = 0.013), indicating that CIDEA downregulation stimulates lipolysis.
To assess whether CIDEA depletion influenced TNF- action, the levels of secreted TNF- protein in cell media from eight of the RNA interference experiments were measured. There was a strong inverse correlation between the levels of secreted TNF- protein and the ratio of CIDEA to GAPDH mRNA (Fig. 3D) (P = 0.0051). The secreted levels of TNF- also correlated with the glycerol release from the same cells (P = 0.03) (graph not shown). In the samples where CIDEA mRNA was inhibited to <10% of control (n = 2), TNF- secretion was increased as much as fourfold.
To determine whether the effect of CIDEA silencing on TNF- secretion was transcriptional or posttranscriptional, we measured the TNF- mRNA levels in the siRNA-treated cells (n = 8). There was no correlation between the ratio of TNF- to GAPDH mRNA and the ratio of CIDEA to GAPDH mRNA (Fig. 4A) (P = 0.29). To assess whether CIDEA downregulation resulted in a selective effect on lipolysis by TNF-, additional measurements were performed in the siRNA-treated cells and incubation media. Adipocytes secrete a number of proteins in addition to TNF- (rev. in 29), among them adiponectin, which is inhibited by TNF- (30). We determined the amount of adiponectin secreted into the media of the cells in the same experiments as in Fig. 3D. The CIDEA mRNA levels in these experiments did not correlate with the amount of adiponectin (P = 0.2, graph not shown).
Because the lipid droplet-associated phosphoprotein perilipin plays an important role in basal lipolysis (31,32), PLIN mRNA levels were assessed in the siRNA-treated cells with the most marked CIDEA downregulation (by 75eC95%, n = 4). In these samples PLIN mRNA levels were 50% lower than in control cells (Fig. 4B) (P = 0.03). The perilipin protein levels were determined by Western blot in four RNA interference experiments. In contrast to mRNA data, there was no significant downregulation of the protein (values not shown).
MCP-1 is upregulated by TNF- in human preadipocytes (33) and 3T3-L1 cells (34). We therefore measured the MCP-1 mRNA levels and secretion of MCP-1 protein in the same RNA interference experiments as in Fig. 3C. The ratio of MCP-1 to GAPDH mRNA did not correlate with the ratio of CIDEA to GAPDH mRNA (Fig. 4C) (P = 0.53). Likewise, the amount of MCP-1 protein secreted into the cell media did not correlate with the ratio of CIDEA to GAPDH mRNA levels (Fig. 4D) (P = 0.38).
CIDEA expression in mice differs qualitatively from that in humans.
To study the species differences in CIDEA expression, we measured the mRNA levels of CIDEA in WAT and BAT from 18 mice of three different strains (C57BL/6J, 129Sv/Pas, and NMRI) that were divided into two groups and fed either normal or cafeteria diet. Data from the C57BL/6J mice on normal diet are shown in Fig. 5AeCB and are representative of all three strains. CIDEA mRNA in WAT was not detectable with SYBR Green-based quantitative real-time PCR but highly so in BAT, confirming earlier studies (16) (Fig. 5A). The mRNA expression of 2-MG in the same samples showed the opposite pattern, with much higher levels in WAT than in BAT (Fig. 5B). Using the more sensitive TaqMan probes, we found 400 times less CIDEA mRNA in WAT as compared with BAT (data not shown). This low level of expression could be nonspecific for fat cells and was not further analyzed. There was a significant diet effect on body weight increase in C57BL/6J mice (P < 0.05) and a borderline significant increase in NMRI mice (Fig. 5C) (P = 0.06). Surprisingly, the body weight increase in 129Sv/Pas mice on normal diet was marginally larger than for the mice on cafeteria diet (Fig. 5C), even though food consumption was equally increased in all three strains (values not shown). There was no effect of the diet or body weight increase in any strain regarding CIDEA mRNA (Fig. 5D).
DISCUSSION
The current study demonstrates that CIDEA is expressed in human white fat cells and that the adipose mRNA levels of CIDEA are markedly influenced by body fat status. This is in clear contrast to mice, where the gene is expressed in brown fat cells only and not influenced by obesity. In human adipose tissue, the expression of CIDEA is downregulated by obesity and normalized after weight reduction. The mRNA levels of CIDEA are inversely correlated to perturbations that are characteristic for the metabolic syndrome: abdominal obesity, enlarged fat cells, insulin resistance (high HOMA index), and increased basal lipolysis (glycerol release). The metabolic syndrome also has other clinical features not measured here, but it is nevertheless obvious that CIDEA mRNA in human WAT is strongly associated with the metabolic syndrome.
Although the RNA interference technique (rev. in 35) has been successfully used in 3T3-L1 preadipocytes (36,37), its usefulness in human preadipocytes has not been reported before. In our experiments, CIDEA mRNA knockdown varied between 20 and 95%, probably because of the interindividual differences of the primary cells used. The effect of CIDEA RNA interference on glycerol release from the preadipocytes correlated with the degree of mRNA downregulation. Our experiments suggest that basal lipolysis in human preadipocytes is under tonic inhibition by CIDEA and that relief of inhibition by mRNA depletion augments lipolysis. This effect most probably involves posttranscriptional modification of TNF- because the release of this prolipolytic cytokine is stimulated by CIDEA downregulation, but there is no corresponding change in the TNF- mRNA levels. CIDEA is apparently a very strong inhibitor of TNF- secretion because adipocytes with >90% CIDEA depletion displayed a fourfold stimulated TNF- release. A similar downregulation of CIDEA also caused a marked decrease in PLIN mRNA but no detectable decrease in the protein amounts of perilipin. This, however, does not exclude perilipin as an indirect target for CIDEA, putatively through TNF-. It may, for example, necessitate a longer time to reduce the amount of protein than mRNA. Furthermore, we (25) and others (38) have previously shown discrepancies between perilipin mRNA and protein levels in human adipose tissue. In addition, CIDEA-TNF- interactions might involve translocation and/or phosphorylation of perilipin, which were not examined here.
CIDEA-TNF- interactions appear to be specific for lipolysis because the mRNA expression and protein secretion of a known target for TNF-, MCP-1, were not influenced by RNA interference. Also, the secretion of the adipose-specific protein adiponectin, whose expression has been shown to be regulated by (39) or associated with (40) TNF- levels, was not affected. At present we do not know why only glycerol release and not secretion of adiponectin or MCP-1 was influenced by increased TNF- release after CIDEA depletion. However, it is possible that glycerol stimulation is an early event, and effects of increased TNF- levels on adiponectin and MCP-1 secretion occur at time points later than 24 h.
The signaling pathways of TNF- to lipolysis are very complex and involve signaling through MAPKs, resulting in downregulation of Gi (inhibitory G-protein -subunit) as well as changes in perilipin phosphorylation and expression (11,24,28,41). In the current study, we have shown that TNF- inhibits CIDEA expression and that this downregulation of CIDEA mRNA is mediated by JNK, one of the MAPKs that is involved in TNF--induced stimulation of lipolysis in human fat cells (24). It is noteworthy that the concentration-dependent relationship for the effect of the JNK inhibitor SP600125 on CIDEA was almost identical to that previously shown on lipolysis using an experimental setting similar to the current study (11). The other two MAPKs that are also active in human fat cells, p44/42 and p38 (11,24,28), do not seem to be involved in regulating CIDEA expression. Our data suggest a reciprocal inhibition of CIDEA and TNF- because CIDEA depletion by siRNA causes an increased secretion of TNF- protein. It is tempting to speculate that the ability of TNF- to inhibit CIDEA is an indirect way for this cytokine to further stimulate lipolysis by counteracting the inhibitory effects of CIDEA on TNF- release. Conversely, the direct way would be through signaling to perilipin and/or other lipolytic regulators.
In the current study, we have only investigated effects of CIDEA depletion. Studies of stable overexpression would require human fat cell lines that are not available. A human fat cell line derived from BAT (42) differs in many qualitative parameters from white adipocytes and can therefore not be used. Transient overexpression is not useful because such ectopic CIDEA expression causes excessive apoptosis in other cell systems (12).
In accordance with previous studies (16), we found that CIDEA is virtually not expressed in WAT in mice. Therefore, CIDEA expression is completely different in mice compared with humans. Humans have easily detectable levels in both WAT and isolated adipocytes. An interesting observation is that the mouse strains displaying low CIDEA levels (C57BL/6J and NMRI) were those that developed diet-induced obesity. It has been suggested that CIDEA may induce obesity through processes in BAT of mice (43), but our results would rather indicate a reverse correlation.
Based on the current results, we propose the following mode of action of CIDEA in human WAT (Fig. 6): CIDEA cross-talks with TNF-, and this has consequences for lipolysis. CIDEA decreases the availability of TNF- by inhibiting cytokine secretion predominately through posttranscriptional mechanisms, which in turn counteracts the ability of TNF- to stimulate lipolysis. TNF- downregulates the expression of CIDEA through signaling via JNK, which in turn increases the availability of TNF- and thereby lipolytic stimulation. This cross-talk seems to be specific for humans. In obesity CIDEA expression is downregulated probably because of increased TNF- action. This elevates the basal lipolytic activity and could be an important contributing factor to elevated circulating fatty acid levels in obesity. Other adipocyte factors responsible for increased basal lipolysis could be additional cytokines and insulin resistance in the fat cells. Because CIDEA is expressed in adipocytes of WAT, it is possible that cell-specific pharmacological agents that either upregulate CIDEA expression or alter the interactions between TNF- and CIDEA (such as JNK inhibitors) could be useful therapeutic agents to decrease fatty acid levels in obese individuals and thereby ameliorate the disadvantageous effects of these lipids.
ACKNOWLEDGMENTS
This study was supported by grants from the Swedish Research Council, the Swedish Diabetes Association, the Swedish Heart and Lung Foundation, the King Gustaf V and Queen Victoria Foundation, the Diabetes Research and Wellness Foundation, the Swedish Cancer Foundation, and EFSD (European Foundation for the Study of Diabetes)/Novo Nordisk Diabetes Research.
FOOTNOTES
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
BAT, brown adipose tissue; CIDEA, cell death-inducing DFFA (DNA fragmentation factor-)-like effector A; HOMA, homeostasis model assessment; JNK, c-Jun NH2-terminal kinase; MAPK, mitogen-activated protein kinase; MCP-1, monocyte chemoattractant protein-1; siRNA, short interfering RNA; TNF-, tumor necrosis factor-; WAT, white adipose tissue
REFERENCES
Hill JO, Melanson EL, Wyatt HT: Dietary fat intake and regulation of energy balance: implications for obesity. J Nutr 130:284SeC288S, 2000
Caterson ID, Gill TP: Obesity: epidemiology and possible prevention. Best Pract Res Clin Endocrinol Metab 16:595eC610, 2002
Stein CJ, Colditz GA: The epidemic of obesity. J Clin Endocrinol Metab 89:2522eC2525, 2004
Carr MC, Brunzell JD: Abdominal obesity and dyslipidemia in the metabolic syndrome: importance of type 2 diabetes and familial combined hyperlipidemia in coronary artery disease risk. J Clin Endocrinol Metab 89:2601eC2607, 2004
Large V, Arner P: Regulation of lipolysis in humans. Pathophysiological modulation in obesity, diabetes, and hyperlipidaemia. Diabetes Metab 24:409eC418, 1998
Blaak EE: Fatty acid metabolism in obesity and type 2 diabetes mellitus. Proc Nutr Soc 62:753eC760, 2003
Hotamisligil GS, Arner P, Caro JF, Atkinson RL, Spiegelman BM: Increased adipose tissue expression of tumor necrosis factor-alpha in human obesity and insulin resistance. J Clin Invest 95:2409eC2415, 1995
Green A, Dobias SB, Walters DJ, Brasier AR: Tumor necrosis factor increases the rate of lipolysis in primary cultures of adipocytes without altering levels of hormone-sensitive lipase. Endocrinology 134:2581eC2588, 1994
Souza SC, Yamamoto MT, Franciosa MD, Lien P, Greenberg AS: BRL 49653 blocks the lipolytic actions of tumor necrosis factor-alpha: a potential new insulin-sensitizing mechanism for thiazolidinediones. Diabetes 47:691eC695, 1998
Hauner H, Petruschke T, Russ M, Rohrig K, Eckel J: Effects of tumour necrosis factor alpha (TNF alpha) on glucose transport and lipid metabolism of newly-differentiated human fat cells in cell culture. Diabetologia 38:764eC771, 1995
Ryden M, Arvidsson E, Blomqvist L, Perbeck L, Dicker A, Arner P: Targets for TNF-alpha-induced lipolysis in human adipocytes. Biochem Biophys Res Commun 318:168eC175, 2004
Inohara N, Koseki T, Chen S, Wu X, Nunez G: CIDE, a novel family of cell death activators with homology to the 45 kDa subunit of the DNA fragmentation factor. EMBO J 17:2526eC2533, 1998
Reed JC, Doctor K, Rojas A, Zapata JM, Stehlik C, Fiorentino L, Damiano J, Roth W, Matsuzawa S, Newman R, Takayama S, Marusawa H, Xu F, Salvesen G, Godzik A: Comparative analysis of apoptosis and inflammation genes of mice and humans. Genome Res 13:1376eC1388, 2003
Danesch U, Hoeck W, Ringold GM: Cloning and transcriptional regulation of a novel adipocyte-specific gene, FSP27: CAAT-enhancer-binding protein (C/EBP) and C/EBP-like proteins interact with sequences required for differentiation-dependent expression. J Biol Chem 267:7185eC7193, 1992
Williams PM, Chang DJ, Danesch U, Ringold GM, Heller RA: CCAAT/enhancer binding protein expression is rapidly extinguished in TA1 adipocyte cells treated with tumor necrosis factor. Mol Endocrinol 6:1135eC1141, 1992
Zhou Z, Yon Toh S, Chen Z, Guo K, Ng CP, Ponniah S, Lin SC, Hong W, Li P: Cidea-deficient mice have lean phenotype and are resistant to obesity. Nat Genet 35:49eC56, 2003
Lofgren P, Hoffstedt J, Ryden M, Thorne A, Holm C, Wahrenberg H, Arner P: Major gender differences in the lipolytic capacity of abdominal subcutaneous fat cells in obesity observed before and after long-term weight reduction. J Clin Endocrinol Metab 87:764eC771, 2002
Emoto M, Nishizawa Y, Maekawa K, Hiura Y, Kanda H, Kawagishi T, Shoji T, Okuno Y, Morii H: Homeostasis model assessment as a clinical index of insulin resistance in type 2 diabetic patients treated with sulfonylureas. Diabetes Care 22:818eC822, 1999
Hoffstedt J, Arvidsson E, Sjolin E, Wahlen K, Arner P: Adipose tissue adiponectin production and adiponectin serum concentration in human obesity and insulin resistance. J Clin Endocrinol Metab 89:1391eC1396, 2004
Hellmer J, Marcus C, Sonnenfeld T, Arner P: Mechanisms for differences in lipolysis between human subcutaneous and omental fat cells. J Clin Endocrinol Metab 75:15eC20, 1992
Arner P: Relationship between intracellular cyclic AMP and lipolysis in human adipose tissue. Acta Med Scand 200:179eC186, 1976
Reynisdottir S, Wahrenberg H, Carlstrom K, Rossner S, Arner P: Catecholamine resistance in fat cells of women with upper-body obesity due to decreased expression of beta 2-adrenoceptors. Diabetologia 37:428eC435, 1994
van Harmelen V, Dicker A, Ryden M, Hauner H, Lonnqvist F, Naslund E, Arner P: Increased lipolysis and decreased leptin production by human omental as compared with subcutaneous preadipocytes. Diabetes 51:2029eC2036, 2002
Ryden M, Dicker A, van Harmelen V, Hauner H, Brunnberg M, Perbeck L, Lonnqvist F, Arner P: Mapping of early signaling events in tumor necrosis factor-alpha-mediated lipolysis in human fat cells. J Biol Chem 277:1085eC1091, 2002
Arvidsson E, Blomqvist L, Ryden M: Depot-specific differences in perilipin mRNA but not protein expression in obesity. J Intern Med 255:595eC601, 2004
Kannisto K, Sutinen J, Korsheninnikova E, Fisher RM, Ehrenborg E, Gertow K, Virkamaki A, Nyman T, Vidal H, Hamsten A, Yki-Jarvinen H: Expression of adipogenic transcription factors, peroxisome proliferator-activated receptor gamma co-activator 1, IL-6 and CD45 in subcutaneous adipose tissue in lipodystrophy associated with highly active antiretroviral therapy. AIDS 17:1753eC1762, 2003
Lin YJ, Harada S, Loten EG, Smith RM, Jarett L: Direct stimulation of immediate-early genes by intranuclear insulin in trypsin-treated H35 hepatoma cells. Proc Natl Acad Sci U S A 89:9691eC9694, 1992
Zhang HH, Halbleib M, Ahmad F, Manganiello VC, Greenberg AS: Tumor necrosis factor-alpha stimulates lipolysis in differentiated human adipocytes through activation of extracellular signal-related kinase and elevation of intracellular cAMP. Diabetes 51:2929eC2935, 2002
Wiecek A, Kokot F, Chudek J, Adamczak M: The adipose tissue—a novel endocrine organ of interest to the nephrologist. Nephrol Dial Transplant 17:191eC195, 2002
Maeda N, Takahashi M, Funahashi T, Kihara S, Nishizawa H, Kishida K, Nagaretani H, Matsuda M, Komuro R, Ouchi N, Kuriyama H, Hotta K, Nakamura T, Shimomura I, Matsuzawa Y: PPAR ligands increase expression and plasma concentrations of adiponectin, an adipose-derived protein. Diabetes 50:2094eC2099, 2001
Souza SC, Muliro KV, Liscum L, Lien P, Yamamoto MT, Schaffer JE, Dallal GE, Wang X, Kraemer FB, Obin M, Greenberg AS: Modulation of hormone-sensitive lipase and protein kinase A-mediated lipolysis by perilipin A in an adenoviral reconstituted system. J Biol Chem 277:8267eC8272, 2002
Tansey JT, Sztalryd C, Gruia-Gray J, Roush DL, Zee JV, Gavrilova O, Reitman ML, Deng CX, Li C, Kimmel AR, Londos C: Perilipin ablation results in a lean mouse with aberrant adipocyte lipolysis, enhanced leptin production, and resistance to diet-induced obesity. Proc Natl Acad Sci U S A 98:6494eC6499, 2001
Wang B, Jenkins JR, Trayhurn P: Expression and secretion of inflammation-related adipokines by human adipocytes differentiated in culture: integrated response to TNF-alpha. Am J Physiol Endocrinol Metab 288:E731eCE740, 2005
Sartipy P, Loskutoff DJ: Monocyte chemoattractant protein 1 in obesity and insulin resistance. Proc Natl Acad Sci U S A 100:7265eC7270, 2003
Hannon GJ, Rossi JJ: Unlocking the potential of the human genome with RNA interference. Nature 431:371eC378, 2004
Xu J, Liao K: Protein kinase B/AKT 1 plays a pivotal role in insulin-like growth factor-1 receptor signaling induced 3T3eCL1 adipocyte differentiation. J Biol Chem 279:35914eC35922, 2004
Mitra P, Zheng X, Czech MP: RNAi-based analysis of CAP, Cbl, and CrkII function in the regulation of GLUT4 by insulin. J Biol Chem 279:37431eC37435, 2004
Wang Y, Sullivan S, Trujillo M, Lee MJ, Schneider SH, Brolin RE, Kang YH, Werber Y, Greenberg AS, Fried SK: Perilipin expression in human adipose tissues: effects of severe obesity, gender, and depot. Obes Res 11:930eC936, 2003
Bruun JM, Lihn AS, Verdich C, Pedersen SB, Toubro S, Astrup A, Richelsen B: Regulation of adiponectin by adipose tissue-derived cytokines: in vivo and in vitro investigations in humans. Am J Physiol Endocrinol Metab 285:E527eCE533, 2003
Kern PA, Di Gregorio GB, Lu T, Rassouli N, Ranganathan G: Adiponectin expression from human adipose tissue: relation to obesity, insulin resistance, and tumor necrosis factor-alpha expression. Diabetes 52:1779eC1785, 2003
Gasic S, Tian B, Green A: Tumor necrosis factor alpha stimulates lipolysis in adipocytes by decreasing Gi protein concentrations. J Biol Chem 274:6770eC6775, 1999
Zilberfarb V, Pietri-Rouxel F, Jockers R, Krief S, Delouis C, Issad T, Strosberg AD: Human immortalized brown adipocytes express functional beta3-adrenoceptor coupled to lipolysis. J Cell Sci 110:801eC807, 1997
Lin SC, Li P: CIDE-A, a novel link between BAT and obesity. Trends Mol Med 10:434eC439, 2004(Elisabet Arvidsson Nordst)