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
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ABSTRACT |
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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.
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RESEARCH DESIGN AND METHODS |
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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 1214 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 412 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 µg 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 ß-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 ß-2 microglobulin (ß2-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. ß2-MG was used as a reference gene except for the TNF-
-treated samples, where ß2-MG in our hands appears to be regulated by TNF-
. Instead, GAPDH was used for RNA interference and TNF-
experiments. ß2-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 ß2-MG or GAPDH, as indicated. To clarify the data not expressed as percent of control, the ratio of CIDEA to ß2-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, Tåstrup, 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 ß2-MG 5'-TGCTGTCTCCATGTTTGATGTATCT-3' and 5'-TCTCTGCTCCCCACCTCTAAGT-3', for mouse CIDEA 5'-AAAGGGACAGAAATGGACAC-3' and 5'-TTGAGACAGCCGAGGAAG-3', and for mouse ß2-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, Wilcoxons signed-rank test, Kruskal-Wallis test, and Spearmans correlation test were used. P < 0.05 was considered significant.
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RESULTS |
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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 ß2-MG mRNA (Fig. 2AD). 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).
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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 3070; BMI 26.5 ± 0.8 kg/m2, range 22.930.2), we performed 24-h incubations with different amounts of siRNA, which resulted in a 2095% 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).
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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. 5AB 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 ß2-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).
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DISCUSSION |
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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.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Address correspondence and reprint requests to Peter Arner, Professor, MD, PhD, Karolinska Institutet, Karolinska University Hospital Huddinge, M63 SE-141 86 Stockholm, Sweden. E-mail: peter.arner{at}medhs.ki.se
Received for publication December 10, 2004 and accepted in revised form March 14, 2005
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
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REFERENCES |
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