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Cidec gene expression in white adipose tissue
Nutrition 28 (2012) 294–299
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Basic nutritional investigation
Intermittent fasting up-regulates Fsp27/Cidec gene expression in white adipose tissue Joanna Karbowska Ph.D. *, Zdzislaw Kochan Ph.D. Department of Biochemistry, Medical University of Gdansk, Gdansk, Poland
a r t i c l e i n f o
a b s t r a c t
Article history: Received 31 March 2011 Accepted 28 June 2011
Objective: Fat-specific protein of 27 kDa (FSP27) is a novel lipid droplet protein that promotes triacylglycerol storage in white adipose tissue (WAT). The regulation of the Fsp27 gene expression in WAT is largely unknown. We investigated the nutritional regulation of FSP27 in WAT. Methods: The effects of intermittent fasting (48 d, eight cycles of 3-d fasting and 3-d refeeding), caloric restriction (48 d), fasting-refeeding (3-d fasting and 3-d refeeding), and fasting (3 d) on mRNA expression of FSP27, peroxisome proliferator-activated receptor g (PPARg2), CCAAT/ enhancer binding protein a (C/EBPa), and M isoform of carnitine palmitoyltransferase 1 (a positive control for PPARg activation) in epididymal WAT and on serum triacylglycerol, insulin, and leptin levels were determined in Wistar rats. We also determined the effects of PPARg activation by rosiglitazone or pioglitazone on FSP27 mRNA levels in primary rat adipocytes. Results: Long-term intermittent fasting, in contrast to other dietary manipulations, significantly upregulated Fsp27 gene expression in WAT. Moreover, in rats subjected to intermittent fasting, serum insulin levels were elevated; PPARg2 and C/EBPa mRNA expression in WAT was increased, and there was a positive correlation of Fsp27 gene expression with PPARg2 and C/EBPa mRNA levels. FSP27 mRNA expression was also increased in adipocytes treated with PPARg agonists. Conclusion: Our study demonstrates that the transcription of the Fsp27 gene in adipose tissue may be induced in response to nutritional stimuli. Furthermore, PPARg2, C/EBPa, and insulin may be involved in the nutritional regulation of FSP27. Thus intermittent fasting, despite lower caloric intake, may promote triacylglycerol deposition in WAT by increasing the expression of genes involved in lipid storage, such as Fsp27. Ó 2012 Elsevier Inc. All rights reserved.
Keywords: Fat-specific protein of 27 kDa PPARg C/EBPa Caloric restriction Nutritional regulation
Introduction Fat-specific protein of 27 kDa (FSP27) belongs to the cell deathinducing DNA fragmentation factor 45-like effector (CIDE) protein family [1,2]. In mammals, three isoforms of CIDE have been described: CIDEA, CIDEB, and FSP27/CIDEC, expressed at high levels in brown adipose tissue (BAT), liver, and white adipose tissue (WAT), respectively [1–5]. Identified previously as mediators of apoptotic cell death [1,2], CIDE proteins have recently been shown to regulate lipid metabolism [4,6]. In the last few years, there has been a rapidly growing interest in the physiologic role of the third member of the CIDE family of proteins, FSP27. It has been demonstrated that FSP27 is a novel lipid droplet protein involved in the formation of unilocular lipid droplets in adipocytes and is required for the efficient accumulation of triacylglycerols in these * Corresponding author. Tel.: þ48 58 3491460; fax: þ48 58 3491465. E-mail address: [email protected] (J. Karbowska). 0899-9007/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.nut.2011.06.009
cells [4,7,8]. Ectopic overexpression of FSP27 in different non-adipose cells also results in spontaneous lipid accumulation [8,9]. FSP27 depletion or deficiency leads to the fragmentation of lipid droplets and enhances basal lipolytic rate in 3T3-L1 adipocytes [3,7,8] and in mice [4]. A homozygous nonsense mutation in CIDEC, the human homolog of the Fsp27 gene, caused partial lipodystrophy and presence of adipocytes with multiple small lipid droplets in the patient’s adipose tissue [10]. Moreover, FSP27 has recently been shown to decrease b-oxidation of fatty acids in adipocytes [7]. These findings indicate that FSP27 plays an important role in lipid metabolism and contributes to efficient energy storage in WAT. Although FSP27 is expressed mainly in white adipose tissue [4], the regulation of Fsp27 gene expression in WAT has not been fully elucidated. It has been shown that the transcription of Fsp27 is induced during differentiation of adipocytes in vitro [4,7,11]. The expression of many adipocyte-specific genes is affected by dietary manipulations, such as fasting and refeeding [12,13]. Since
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Magnusson et al. demonstrated that treatment of morbidly obese patients with a very low calorie diet (VLCD) down-regulates CIDEC expression in their subcutaneous adipose tissue [3], and, very recently, Ito et al. reported an increase in CIDEC mRNA levels in human adipocytes treated with insulin [14], it appears that Fsp27/CIDEC gene expression is regulated by nutritional signals. However, to date there are no reported studies to our knowledge on the Fsp27 gene response to different nutritional conditions. To investigate the nutritional regulation of FSP27, we determined the effects of intermittent fasting, caloric restriction, fastingrefeeding, and fasting on Fsp27 gene expression in rat epididymal WAT. We demonstrate here that intermittent fasting strongly up-regulates the expression of the Fsp27 gene in adipose tissue, and that peroxisome proliferator-activated receptor g (PPARg), CCAAT/enhancer binding protein a (C/EBPa), and insulin are involved in this regulation. Materials and methods Animals Male Wistar rats weighing 304.2 19.8 g at the start of the experiment were housed individually in wire-mesh cages and maintained at 22 C in an animal facility with a 12:12-h light-dark cycle (lights on at 8:00 a.m.). All animals were given ad libitum access to water and fed a commercial rodent diet. The composition of the diet has been described in detail previously [15]. The rats were randomly assigned to five experimental groups: fasted rats (F, n ¼ 5) were fed ad libitum for 45 d and then deprived of food for 3 d, fasted and refed rats (FR, n ¼ 5) were fed ad libitum for 42 d and then subjected to 3-d food deprivation and subsequently refed for 3 d, intermittently fasted rats (IF, n ¼ 5) were subjected to eight 6-d cycles of food deprivation (3 d) and refeeding (3 d), caloric restricted rats (CR, n ¼ 5) received the same amount of food as consumed by IF rats over the preceding 6-d cycle of food deprivation and refeeding, i.e., with daily food intake reduced to 80% of control (ad libitum) levels—both intermittent fasting and caloric restriction lasted for 48 d, and control rats (C, n ¼ 10) that were given ad libitum access to food. The rats were killed by cervical dislocation between 8:00 and 10:00 a.m. Their trunk blood was collected and used for biochemical assays. Epididymal adipose tissue was taken from each experimental group. Tissue specimens were immediately frozen in liquid nitrogen and were stored at 80 C until required for analysis. The experimental protocol was approved by the Local Ethics Committee for Animal Experimentation. Biochemical assays Serum insulin and leptin levels were measured with radioimmunoassay kits (Linco Research, St. Charles, MO, USA), and triacylglycerol concentrations were determined by an enzymatic method (Roche Diagnostics, Mannheim, Germany). Assays were performed according to the manufacturers’ instructions. Isolation of primary rat adipocytes Mature adipocytes were prepared from epididymal fat pads of 2- to 4-mo-old male Wistar rats by collagenase digestion as previously described by Rodbell [16] with slight modification. Dissected tissue samples were minced with scissors and digested with 0.5 mg/mL collagenase (type II; Sigma, St. Louis, MO, USA) in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 5% bovine serum albumin for 60 min at 37 C with continuous shaking (150 rpm). The resulting cell suspension was filtered through a nylon mesh. The adipocytes were subsequently washed three times and resuspended in DMEM containing 25 mM glucose and 10% fetal bovine serum. Incubation of primary rat adipocytes with PPARg agonists Freshly isolated mature adipocytes were incubated in 1 mL (5 105 cells/mL) of DMEM supplemented with 25 mM glucose and 10% fetal bovine serum, and treated with PPARg agonists, either rosiglitazone (10 mM) or pioglitazone (10 mM) (Enzo Life Sciences, Farmingdale, NY, USA), for 24 h at 37 C, 5% CO2. RNA isolation and expression analysis by real-time RT-PCR Total RNA was extracted from adipose tissue samples as previously described [12]. Isolated primary adipocytes after incubation with PPARg agonists were disrupted by adding 1 mL of QIAzol Lysis Reagent (Qiagen, Hilden, Germany) and homogenized for 1 min. Total RNA was then extracted with the RNeasy Lipid
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Tissue Mini Kit (Qiagen), according to the manufacturer’s instructions. The concentration and integrity of RNA samples were determined using the Experion Automated Electrophoresis System and RNA StdSens Analysis Kit (Bio-Rad, Hercules, CA, USA). mRNA expression of FSP27, PPARg2, C/EBPa, M isoform of carnitine palmitoyltransferase 1 (MCPT1), and acidic ribosomal phosphoprotein P0 (36B4) was quantified by real-time reverse transcription-polymerase chain reaction (RT-PCR) using the Chromo4 Real-Time PCR Detection System (Bio-Rad). Primers were designed with Sequence Analysis software package (Informagen, Newington, USA) from gene sequences obtained from Ensembl Genome Browser (www.ensembl.org). Primer sequences were as follows: FSP27, forward 50 -GTG TTA GCA CCG CAG ATC G-30 , reverse 50 -CAC GAT TGT GCC ATC TTC C-30 ; PPARg2, forward 50 -GAT TTG AAA GAA GCT GTG AAC C-30 , reverse 50 -GAA ATC AAC CGT GGT AAA GG-30 ; C/EBPa, forward 50 -AAG TCG GTG GAT AAG AAC AGC-30 , reverse 50 -TCA ACT CCA ACA CCT TCT GC-30 ; MCPT1, forward 50 -CAT GGT GAA CAG CAA CTA TTA CG-30 , reverse 50 -CAT CTG GTA GGA GCA CAT GG-30 ; 36B4, forward 50 GCG ACC TGG AAG TCC AAC TAC-30 , reverse 50 -TCT GCT GCA TCT GCT TGG-30 . One microgram of total RNA was converted to cDNA using the iScript cDNA Synthesis Kit (Bio-Rad). The cDNA (1% of RT reaction) was amplified by PCR using gene-specific primer pairs (0.9 mmol/L of each primer in a 20 mL reaction mixture) and iQ SYBR Green Supermix (Bio-Rad). The samples were incubated at 95 C for 5 min followed by 40 cycles of 95 C for 20 s, 55 C for 20 s, and 72 C for 40 s. Negative controls with no template cDNA (without RT) were included in each PCR. All samples were run in triplicate. SYBR Green I fluorescence was used to detect the accumulation of the PCR products and to assess their expression levels. The relative abundance of gene transcripts was determined on the basis of Ct values (Ct, threshold cycle) using the DDCt formula [17]. 36B4 was used as an endogenous reference gene to validate the Ct readings of RT-PCR and to adjust for any potential unaccounted variation or bias. Specificity of amplified PCR products was assessed by performing a melting curve analysis. PCR data were analyzed with the Opticon Monitor 3 software (Bio-Rad) and Excel (Microsoft). The results are expressed in arbitrary units, and the mean mRNA expression in control samples is set at one. Statistical analysis The results, expressed in arbitrary units, are presented as means standard error of the mean (SEM). The statistical significance of differences found between groups was assessed by one-way analysis of variance. An unpaired Student’s t test or a Kruskal-Wallis test was used to compare the groups. P < 0.05 was considered as a significant difference. Statistical analysis was performed using GraphPad Prism software (GraphPad Software, San Diego, CA, USA).
Results Effect of dietary manipulations on body weight, epididymal WAT mass, serum triacylglycerol, insulin, and leptin levels At the end of the experiment, intermittently fasted and caloric restricted rats weighed less than controls; however, changes in body weight of CR rats were less pronounced (Table 1). Moreover, dietary manipulations led to a decrease in epididymal WAT mass in rats from F, FR, IF, and CR groups when compared to the control group. The most significant reduction in epididymal WAT mass was observed in rats subjected to intermittent fasting. Similarly, serum leptin concentrations were significantly decreased in F, FR, IF, and CR as compared to control rats. Three days of food deprivation decreased circulating triacylglycerol and insulin levels in F rats relative to controls. Triacylglycerol concentrations were also lower in serum of FR and IF rats. In contrast, serum insulin levels were significantly increased after fasting-refeeding and intermittent fasting as compared to the control group. Intermittent fasting significantly up-regulates Fsp27 gene expression in rat epididymal adipose tissue Fsp27 expression was highly induced in rat WAT in response to eight cycles of food deprivation and refeeding. In IF rats, the levels of FSP27 mRNA were more than two-fold higher than in control rats (Fig. 1A). Other dietary manipulations, such as caloric restriction, fasting-refeeding, and fasting, had no effect on the expression of Fsp27 in rat epididymal adipose tissue.
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Table 1 Body weight, epididymal WAT mass, serum triacylglycerol, insulin, and leptin levels in control rats (C) and rats subjected to fasting (F), fasting and refeeding (FR), intermittent fasting (IF), and caloric restriction (CR) C Body weight [g] WAT mass [g] Serum triacylglycerols [mg/dL] Serum insulin [ng/mL] Serum leptin [ng/mL]
447.5 4.45 166.9 0.56 3.06
F
19.9a 0.34a 23.8a 0.19a 0.37a
414.9 3.28 57.0 0.09 1.63
FR
20.5a 0.39b 10.3b 0.01b 0.11b
426.2 3.48 69.8 2.14 1.86
IF
23.1a 0.40b 3.84b 0.87c 0.13b
325.9 2.37 59.21 4.55 2.13
CR
16.9b 0.14c 3.27b 0.33d 0.12b
375.9 3.72 151.3 0.86 2.05
17.2b 0.39b 10.9a 0.25a 0.20b
Values in the same row with different superscript letters are significantly different (P < 0.05)
Effect of dietary manipulations on PPARg2 mRNA levels in rat epididymal adipose tissue To elucidate whether PPARg2 is implicated in the nutritional regulation of Fsp27 gene expression in adipose tissue, PPARg2 mRNA levels were determined by real-time RT-PCR in epididymal WAT of rats subjected to intermittent fasting, caloric restriction, fasting-refeeding, and fasting. Intermittent fasting markedly up-regulated PPARg2 expression in rat WAT. In IF rats, PPARg2 mRNA levels were approximately two-fold higher than in the control group (Fig. 1B). Moreover, the expression levels of MCPT1, a known PPARg target gene in rodent white fat, were substantially increased in WAT of IF rats (Fig. 1C). In contrast to intermittent fasting, other dietary manipulations had no impact on PPARg2 mRNA expression in rat WAT. Furthermore, there was a significant positive correlation between the levels of FSP27 and PPARg2 mRNAs in adipose tissue (Fig. 2A). PPARg agonists stimulate the expression of FSP27 mRNA in isolated primary adipocytes To determine if PPARg activation might be responsible for the up-regulation of FSP27 in rat WAT, we isolated adipocytes from epididymal WAT of control rats and incubated them for 24 h with highly selective PPARg agonists, either rosiglitazone or pioglitazone. Treatment of primary rat adipocytes with PPARg agonists resulted in a significant increase in FSP27 mRNA levels (Fig. 3A). The expression of the MCPT1 encoding gene, used as a positive control for PPARg activation, was also increased in rat epididymal adipocytes treated with rosiglitazone or pioglitazone (Fig. 3B). Intermittent fasting increases C/EBPa mRNA expression in rat epididymal adipose tissue We next investigated whether C/EBPa may be involved in the nutritional regulation of Fsp27 gene expression in rat WAT. The mRNA expression levels of C/EBPa were quantified by real-time RT-PCR in epididymal adipose tissue of rats subjected to intermittent fasting, caloric restriction, fasting-refeeding, and fasting. C/EBPa mRNA levels in IF rats were markedly increased, approximately three-fold when compared to control rats (Fig. 1D). In contrast, C/EBPa mRNA expression was not altered by other dietary manipulations. Moreover, a strong positive correlation was found between Fsp27 gene expression and C/EBPa mRNA levels in rat WAT (Fig. 2B). Discussion Fsp27 was originally discovered as the adipocyte-specific gene, highly expressed only in fully differentiated adipocytes [11]. FSP27, and its human homolog, CIDEC, is expressed almost
exclusively in white adipose tissue [3,4,18]. In mice, the highest level of expression of the Fsp27 gene was found in epididymal WAT [18]. In the present work we investigated the effect of dietary manipulations, such as intermittent fasting, caloric restriction, fasting-refeeding, and fasting, on the expression of Fsp27 in rat epididymal adipose tissue. We found that intermittent fasting, in contrast to other dietary manipulations, up-regulates Fsp27 gene expression. To our knowledge, this is the first report demonstrating the induction of the Fsp27 gene transcription in response to nutritional stimuli. In cultured adipocytes, the Fsp27 gene was induced during adipogenesis [4,7,11]. The process of adipocyte differentiation is governed by two transcription factors, PPARg and C/EBPa [19]. Induced early in adipogenesis, PPARg acts as a crucial regulator of many adipocyte-specific genes and triggers the adipocyte differentiation program [19]. C/EBPa is induced relatively late during adipogenesis and is implicated in the maintenance of the adipocyte phenotype [19]. As PPARg and C/EBPa orchestrate the expression of several genes in adipocytes, we hypothesised that these transcription factors may be involved in the up-regulation of Fsp27 gene expression in WAT during intermittent fasting. PPARg2 is the isoform of PPAR restricted to adipose tissue [20]; thus, we measured PPARg2 and C/EBPa gene expression in epididymal WAT of rats subjected to intermittent fasting, caloric restriction, fasting-refeeding, and fasting. We found that mRNA levels of both transcription factors were remarkably increased in IF rats. It has been shown previously that PPARg2 gene expression in adipose tissue is not affected by diet composition [21]. However, adipose expression of PPARg2 is down-regulated by fasting and restored by refeeding [13,20]. Our studies demonstrate that PPARg2 is strongly up-regulated in WAT by multiple cycles of fasting and refeeding (intermittent fasting). Moreover, the induction of PPARg2 gene expression in white fat of IF rats was accompanied by activation of this transcription factor. In WAT of rats subjected to intermittent fasting the expression levels of MCPT1, previously identified as a PPARg target gene in rodent white fat [22], were significantly increased, indicating the enhanced transcriptional activity of PPARg. Furthermore, a significant positive correlation was found between PPARg2 and FSP27 mRNA levels in rat adipose tissue, suggesting that PPARg2 is involved in the nutritional regulation of FSP27 in WAT. Based on pharmacologic studies, one can assume that Fsp27 is a PPARg target gene. Recently, it has been demonstrated that PPARg binds directly to the PPRE element localized in the Fsp27 promoter region and increases Fsp27 promoter activity in adipocytes [9,23]. Treatment with PPARg agonist rosiglitazone increased FSP27 mRNA levels in WAT of obese and lean mice [24]. Furthermore, in vitro studies have shown that FSP27 mRNA expression in 3T3-L1 adipocytes was elevated after PPARg agonist treatment [4,23]. However, in studies published by Puri et al. rosiglitazone treatment of mature 3T3-L1 adipocytes did not influence Fsp27 gene expression [24]. These results imply that FSP27 response to
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Fig. 2. Relationship between FSP27 and PPARg2 (A) and C/EBPa (B) mRNA levels in rat epididymal WAT. Pearson’s correlation was used to determine the relationship between variables.
Fig. 1. mRNA expression levels of FSP27 (A), PPARg2 (B), MCPT1 (C), and C/EBPa (D) in epididymal WAT of control rats (C) and rats subjected to fasting (F), fasting and refeeding (FR), intermittent fasting (IF), and caloric restriction (CR). Values with different superscript letters are significantly different (P < 0.05).
PPARg agonists may be affected by the degree of differentiation of 3T3-L1 cells. Therefore, we used mature adipocytes isolated from epididymal WAT of control rats to elucidate whether PPARg may contribute to the nutritional regulation of FSP27 during intermittent fasting. In primary rat adipocytes, activation of PPARg by rosiglitazone or pioglitazone confirmed by the up-regulation of MCPT1 gene expression, led to a substantial increase in FSP27 mRNA levels, indicating that PPARg may play a significant role in the transcriptional activation of the Fsp27 gene in response to intermittent fasting. This is the first report, to our knowledge, demonstrating the induction of Fsp27 gene expression by activators of PPARg in primary adipocytes. C/EBPa was also significantly up-regulated in WAT of IF rats and its mRNA levels correlated well with the expression of Fsp27. Because interaction of C/EBPa with the Fsp27 promoter has been previously demonstrated in cultured adipocytes [11], we propose that C/EBPa is implicated in the intermittent fasting-dependent induction of Fsp27 gene expression. We found that serum insulin levels were substantially increased in rats subjected to intermittent fasting as compared to other groups. In previous studies, no association has been found, to our knowledge, between CIDEC mRNA expression in human subcutaneous adipose tissue and resting plasma insulin levels [7]. However, in vitro experiments have shown that insulin may be directly involved in the regulation of Fsp27 gene expression. In 3T3L1 adipocytes, insulin increased FSP27 mRNA levels [6]. Moreover, Ito et al. demonstrated recently that insulin was able to induce the expression of CIDEC in human preadipocytes differentiated in vitro and to increase CIDEC mRNA levels in a time- and dose-dependent manner [14]. Because insulin regulates the transcription of many adipocyte-specific genes in vivo, we assume that it is a positive regulator of Fsp27 expression during intermittent fasting.
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Fig. 3. Effect of PPARg agonists, rosiglitazone (Rosi) and pioglitazone (Pio), on FSP27 (A) and MCPT1 (B) mRNA levels in primary adipocytes isolated from epididymal WAT of control rats. Values represent means SEM of n ¼ 3 independent experiments (**P < 0.01 versus vehicle treated).
In this study, rats subjected to long-term dietary restriction, either IF or CR, gained less weight than control rats and showed reduced WAT mass, which was reflected by lower circulating leptin levels. Total calorie intake was the same between IF and CR groups; however, the reduction in WAT mass and serum triacylglycerol levels was greater in IF rats than in CR rats. This difference may result from increased fatty acid oxidation in WAT of rats subjected to intermittent fasting. Consistent with this is our finding that the gene encoding MCPT1, a key enzyme involved in b-oxidation of fatty acids in adipocytes, is up-regulated in white fat of IF rats. We have previously shown that multiple cycles of fasting and refeeding significantly increase mRNA levels and activities of lipogenic enzymes, thus enhancing lipogenesis in rat WAT [12,15,25]. The present study demonstrates that intermittent fasting may promote lipid deposition in adipose tissue by increasing the expression of FSP27, a protein involved in triacylglycerol accumulation within lipid droplets. Despite this, rats fed ad libitum after fasting-refeeding cycles remained lean and weighed less than controls [15,25]. Thus, it seems unlikely that overexpression of FSP27 in adipose tissue may contribute to the development of obesity. This is consistent with studies in humans showing that FSP27/CIDEC mRNA expression in subcutaneous adipose tissue is either uncorrelated or negatively correlated with body mass index and total fat mass [3,7]. In contrast to intermittent fasting, other dietary manipulations did not affect Fsp27 gene expression in rat adipose tissue. It is somewhat surprising that
FSP27 mRNA levels were not changed in CR rats, which received the same amount of food as IF rats, i.e., their daily food intake was reduced to 80% of control levels. These results indicate that intermittent fasting, but not a reduction in caloric intake per se, is responsible for the up-regulation of Fsp27 expression in WAT. Magnusson et al. recently reported a decrease in CIDEC mRNA levels in subcutaneous adipose tissue of morbidly obese patients undergoing treatment with a very low calorie diet [3]. However, it should be noted that these patients received less than 25% of the recommended daily caloric intake during a 16-wk treatment period. It suggests that suppression of the Fsp27 gene in adipose tissue may require a long-term and severe reduction in food intake. More modest reduction in caloric intake, as in CR rats, appears to have a minor or no effect on FSP27 mRNA levels in WAT. Taken together, these observations indicate that the regulation of Fsp27 gene expression associated with caloric restriction needs further investigation. Recent findings indicate that FSP27 is required for optimal energy storage in WAT and plays an important role in wholebody energy homeostasis. FSP27-deficient mice are protected from diet-induced obesity and insulin resistance [4,26]. However, the cellular and molecular mechanisms underlying the enhanced insulin sensitivity in FSP27-knockout mice remain largely unknown. Furthermore, the role of FSP27/CIDEC in regulating insulin sensitivity has not been fully established. In studies of obese and non-obese persons published by Keller et al., there was no association between CIDEC expression in subcutaneous adipose tissue of participants and HOMA index, or resting plasma insulin and glucose levels [7]. On the other hand, Puri et al. have found that CIDEC mRNA levels are lower in omental and subcutaneous adipose tissue of obese, insulin-resistant subjects than in insulin-sensitive subjects matched for body mass index [24]. Moreover, it has been recently reported that a loss-of-function mutation in CIDEC is associated with insulin-resistant diabetes in a female patient [10]. These latter findings suggest that reduced expression of CIDEC in adipose tissue may contribute to the pathogenesis of insulin resistance. In view of these observations, it is likely that up-regulation of FSP27 in response to intermittent fasting may enhance lipid storage in WAT, thus improving whole-body insulin sensitivity. Recent studies demonstrating the beneficial effects of intermittent energy restriction on insulin resistance in humans [27] seem to confirm this hypothesis.
Conclusion Long-term intermittent fasting, but not caloric restriction or any other dietary manipulation, significantly up-regulates Fsp27 expression in adipose tissue. Furthermore, PPARg, C/EBPa, and insulin are involved in the nutritional regulation of the Fsp27 gene in WAT. Up-regulation of FSP27 may have promoted lipid deposition in WAT after intermittent fasting; however, it did not result in the development of obesity. Further studies are needed to determine the effect of intermittent fasting-induced overexpression of FSP27 in WAT on energy homeostasis and insulin sensitivity.
Acknowledgments This work was supported by the Ministry of Science and Higher Education (2 P05A 019 30) and the Medical University of Gdansk (W-84 and ST-41).
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References [1] 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 1998;17:2526–33. [2] Liang L, Zhao M, Xu Z, Yokoyama KK, Li T. Molecular cloning and characterization of CIDE-3, a novel member of the cell-death-inducing DNA-fragmentation-factor (DFF45)-like effector family. Biochem J 2003;370:195–203. [3] Magnusson B, Gummesson A, Glad CA, Goedecke JH, Jernas M, Lystig TC, et al. Cell death-inducing DFF45-like effector C is reduced by caloric restriction and regulates adipocyte lipid metabolism. Metabolism 2008;57:1307–13. [4] Nishino N, Tamori Y, Tateya S, Kawaguchi T, Shibakusa T, Mizunoya W, et al. FSP27 contributes to efficient energy storage in murine white adipocytes by promoting the formation of unilocular lipid droplets. J Clin Invest 2008;118:2808–21. [5] Zhou Z, Toh SY, Chen Z, Guo K, Ng CP, Ponniah S, et al. Cidea-deficient mice have lean phenotype and are resistant to obesity. Nat Genet 2003;35:49–56. [6] Kim JY, Liu K, Zhou S, Tillison K, Wu Y, Smas CM. Assessment of fat-specific protein 27 in the adipocyte lineage suggests a dual role for FSP27 in adipocyte metabolism and cell death. Am J Physiol Endocrinol Metab 2008;294:E654–67. [7] Keller P, Petrie JT, De Rose P, Gerin I, Wright WS, Chiang SH, et al. Fat-specific protein 27 regulates storage of triacylglycerol. J Biol Chem 2008;283:14355–65. [8] Puri V, Konda S, Ranjit S, Aouadi M, Chawla A, Chouinard M, et al. Fatspecific protein 27, a novel lipid droplet protein that enhances triglyceride storage. J Biol Chem 2007;282:34213–8. [9] Matsusue K, Kusakabe T, Noguchi T, Takiguchi S, Suzuki T, Yamano S, et al. Hepatic steatosis in leptin-deficient mice is promoted by the PPARg target gene Fsp27. Cell Metab 2008;7:302–11. [10] Rubio-Cabezas O, Puri V, Murano I, Saudek V, Semple RK, Dash S, et al. Partial lipodystrophy and insulin resistant diabetes in a patient with a homozygous nonsense mutation in CIDEC. EMBO Mol Med 2009;1:280–7. [11] Danesch U, Hoeck W, Ringold GM. Cloning and transcriptional regulation of a novel adipocyte-specific gene, FSP27. CCAAT-enhancer-binding protein (C/EBP) and C/EBP-like proteins interact with sequences required for differentiation-dependent expression. J Biol Chem 1992;267:7185–93. [12] Karbowska J, Kochan Z, Swierczynski J. Increase of lipogenic enzyme mRNA levels in rat white adipose tissue after multiple cycles of starvationrefeeding. Metabolism 2001;50:734–8. [13] Palou M, Sanchez J, Priego T, Rodriguez AM, Pico C, Palou A. Regional differences in the expression of genes involved in lipid metabolism in adipose tissue in response to short- and medium-term fasting and refeeding. J Nutr Biochem 2010;21:23–33.
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[14] Ito M, Nagasawa M, Hara T, Ide T, Murakami K. Differential roles of CIDEA and CIDEC in insulin-induced anti-apoptosis and lipid droplet formation in human adipocytes. J Lipid Res 2010;51:1676–84. [15] Kochan Z, Karbowska J, Swierczynski J. Unusual increase of lipogenesis in rat white adipose tissue after multiple cycles of starvation-refeeding. Metabolism 1997;46:10–7. [16] Rodbell M. Metabolism of isolated fat cells. I. Effects of hormones on glucose metabolism and lipolysis. J Biol Chem 1964;239:375–80. [17] Winer J, Jung CK, Shackel I, Williams PM. Development and validation of real-time quantitative reverse transcriptase-polymerase chain reaction for monitoring gene expression in cardiac myocytes in vitro. Anal Biochem 1999;270:41–9. [18] Puri V, Virbasius JV, Guilherme A, Czech MP. RNAi screens reveal novel metabolic regulators: RIP140, MAP4k4 and the lipid droplet associated fat specific protein (FSP) 27. Acta Physiol 2008;192:103–15. [19] Rosen ED, Hsu CH, Wang X, Sakai S, Freeman MW, Gonzalez FJ, et al. C/EBPa induces adipogenesis through PPARg: a unified pathway. Genes Dev 2002;16:22–6. [20] Escher P, Braissant O, Basu-Modak S, Michalik L, Wahli W, Desvergne B. Rat PPARs: quantitative analysis in adult rat tissues and regulation in fasting and refeeding. Endocrinology 2001;142:4195–202. [21] Rousseau V, Becker DJ, Ongemba LN, Rahier J, Henquin JC, Brichard SM. Developmental and nutritional changes of ob and PPARg2 gene expression in rat white adipose tissue. Biochem J 1997;321:451–6. [22] Wilson-Fritch L, Nicoloro S, Chouinard M, Lazar MA, Chui PC, Leszyk J, et al. Mitochondrial remodeling in adipose tissue associated with obesity and treatment with rosiglitazone. J Clin Invest 2004;114:1281–9. [23] Kim YJ, Cho SY, Yun CH, Moon YS, Lee TR, Kim SH. Transcriptional activation of Cidec by PPARg2 in adipocyte. Biochem Biophys Res Commun 2008;377:297–302. [24] Puri V, Ranjit S, Konda S, Nicoloro SM, Straubhaar J, Chawla A, et al. Cidea is associated with lipid droplets and insulin sensitivity in humans. Proc Natl Acad Sci USA 2008;105:7833–8. [25] Kochan Z, Karbowska J, Swierczynski J. The effects of weight cycling on serum leptin levels and lipogenic enzyme activities in adipose tissue. J Physiol Pharmacol 2006;57(suppl 6):115–27. [26] Toh SY, Gong J, Du G, Li JZ, Yang S, Ye J, et al. Up-regulation of mitochondrial activity and acquirement of brown adipose tissue-like property in the white adipose tissue of fsp27 deficient mice. PLoS One 2008;3:e2890. [27] Harvie MN, Pegington M, Mattson MP, Frystyk J, Dillon B, Evans G, et al. The effects of intermittent or continuous energy restriction on weight loss and metabolic disease risk markers: a randomized trial in young overweight women. Int J Obes 2011;35:714–27.
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Basic nutritional investigation
Intermittent fasting up-regulates Fsp27/Cidec gene expression in white adipose tissue Joanna Karbowska Ph.D. *, Zdzislaw Kochan Ph.D. Department of Biochemistry, Medical University of Gdansk, Gdansk, Poland
a r t i c l e i n f o
a b s t r a c t
Article history: Received 31 March 2011 Accepted 28 June 2011
Objective: Fat-specific protein of 27 kDa (FSP27) is a novel lipid droplet protein that promotes triacylglycerol storage in white adipose tissue (WAT). The regulation of the Fsp27 gene expression in WAT is largely unknown. We investigated the nutritional regulation of FSP27 in WAT. Methods: The effects of intermittent fasting (48 d, eight cycles of 3-d fasting and 3-d refeeding), caloric restriction (48 d), fasting-refeeding (3-d fasting and 3-d refeeding), and fasting (3 d) on mRNA expression of FSP27, peroxisome proliferator-activated receptor g (PPARg2), CCAAT/ enhancer binding protein a (C/EBPa), and M isoform of carnitine palmitoyltransferase 1 (a positive control for PPARg activation) in epididymal WAT and on serum triacylglycerol, insulin, and leptin levels were determined in Wistar rats. We also determined the effects of PPARg activation by rosiglitazone or pioglitazone on FSP27 mRNA levels in primary rat adipocytes. Results: Long-term intermittent fasting, in contrast to other dietary manipulations, significantly upregulated Fsp27 gene expression in WAT. Moreover, in rats subjected to intermittent fasting, serum insulin levels were elevated; PPARg2 and C/EBPa mRNA expression in WAT was increased, and there was a positive correlation of Fsp27 gene expression with PPARg2 and C/EBPa mRNA levels. FSP27 mRNA expression was also increased in adipocytes treated with PPARg agonists. Conclusion: Our study demonstrates that the transcription of the Fsp27 gene in adipose tissue may be induced in response to nutritional stimuli. Furthermore, PPARg2, C/EBPa, and insulin may be involved in the nutritional regulation of FSP27. Thus intermittent fasting, despite lower caloric intake, may promote triacylglycerol deposition in WAT by increasing the expression of genes involved in lipid storage, such as Fsp27. Ó 2012 Elsevier Inc. All rights reserved.
Keywords: Fat-specific protein of 27 kDa PPARg C/EBPa Caloric restriction Nutritional regulation
Introduction Fat-specific protein of 27 kDa (FSP27) belongs to the cell deathinducing DNA fragmentation factor 45-like effector (CIDE) protein family [1,2]. In mammals, three isoforms of CIDE have been described: CIDEA, CIDEB, and FSP27/CIDEC, expressed at high levels in brown adipose tissue (BAT), liver, and white adipose tissue (WAT), respectively [1–5]. Identified previously as mediators of apoptotic cell death [1,2], CIDE proteins have recently been shown to regulate lipid metabolism [4,6]. In the last few years, there has been a rapidly growing interest in the physiologic role of the third member of the CIDE family of proteins, FSP27. It has been demonstrated that FSP27 is a novel lipid droplet protein involved in the formation of unilocular lipid droplets in adipocytes and is required for the efficient accumulation of triacylglycerols in these * Corresponding author. Tel.: þ48 58 3491460; fax: þ48 58 3491465. E-mail address: [email protected] (J. Karbowska). 0899-9007/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.nut.2011.06.009
cells [4,7,8]. Ectopic overexpression of FSP27 in different non-adipose cells also results in spontaneous lipid accumulation [8,9]. FSP27 depletion or deficiency leads to the fragmentation of lipid droplets and enhances basal lipolytic rate in 3T3-L1 adipocytes [3,7,8] and in mice [4]. A homozygous nonsense mutation in CIDEC, the human homolog of the Fsp27 gene, caused partial lipodystrophy and presence of adipocytes with multiple small lipid droplets in the patient’s adipose tissue [10]. Moreover, FSP27 has recently been shown to decrease b-oxidation of fatty acids in adipocytes [7]. These findings indicate that FSP27 plays an important role in lipid metabolism and contributes to efficient energy storage in WAT. Although FSP27 is expressed mainly in white adipose tissue [4], the regulation of Fsp27 gene expression in WAT has not been fully elucidated. It has been shown that the transcription of Fsp27 is induced during differentiation of adipocytes in vitro [4,7,11]. The expression of many adipocyte-specific genes is affected by dietary manipulations, such as fasting and refeeding [12,13]. Since
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Magnusson et al. demonstrated that treatment of morbidly obese patients with a very low calorie diet (VLCD) down-regulates CIDEC expression in their subcutaneous adipose tissue [3], and, very recently, Ito et al. reported an increase in CIDEC mRNA levels in human adipocytes treated with insulin [14], it appears that Fsp27/CIDEC gene expression is regulated by nutritional signals. However, to date there are no reported studies to our knowledge on the Fsp27 gene response to different nutritional conditions. To investigate the nutritional regulation of FSP27, we determined the effects of intermittent fasting, caloric restriction, fastingrefeeding, and fasting on Fsp27 gene expression in rat epididymal WAT. We demonstrate here that intermittent fasting strongly up-regulates the expression of the Fsp27 gene in adipose tissue, and that peroxisome proliferator-activated receptor g (PPARg), CCAAT/enhancer binding protein a (C/EBPa), and insulin are involved in this regulation. Materials and methods Animals Male Wistar rats weighing 304.2 19.8 g at the start of the experiment were housed individually in wire-mesh cages and maintained at 22 C in an animal facility with a 12:12-h light-dark cycle (lights on at 8:00 a.m.). All animals were given ad libitum access to water and fed a commercial rodent diet. The composition of the diet has been described in detail previously [15]. The rats were randomly assigned to five experimental groups: fasted rats (F, n ¼ 5) were fed ad libitum for 45 d and then deprived of food for 3 d, fasted and refed rats (FR, n ¼ 5) were fed ad libitum for 42 d and then subjected to 3-d food deprivation and subsequently refed for 3 d, intermittently fasted rats (IF, n ¼ 5) were subjected to eight 6-d cycles of food deprivation (3 d) and refeeding (3 d), caloric restricted rats (CR, n ¼ 5) received the same amount of food as consumed by IF rats over the preceding 6-d cycle of food deprivation and refeeding, i.e., with daily food intake reduced to 80% of control (ad libitum) levels—both intermittent fasting and caloric restriction lasted for 48 d, and control rats (C, n ¼ 10) that were given ad libitum access to food. The rats were killed by cervical dislocation between 8:00 and 10:00 a.m. Their trunk blood was collected and used for biochemical assays. Epididymal adipose tissue was taken from each experimental group. Tissue specimens were immediately frozen in liquid nitrogen and were stored at 80 C until required for analysis. The experimental protocol was approved by the Local Ethics Committee for Animal Experimentation. Biochemical assays Serum insulin and leptin levels were measured with radioimmunoassay kits (Linco Research, St. Charles, MO, USA), and triacylglycerol concentrations were determined by an enzymatic method (Roche Diagnostics, Mannheim, Germany). Assays were performed according to the manufacturers’ instructions. Isolation of primary rat adipocytes Mature adipocytes were prepared from epididymal fat pads of 2- to 4-mo-old male Wistar rats by collagenase digestion as previously described by Rodbell [16] with slight modification. Dissected tissue samples were minced with scissors and digested with 0.5 mg/mL collagenase (type II; Sigma, St. Louis, MO, USA) in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 5% bovine serum albumin for 60 min at 37 C with continuous shaking (150 rpm). The resulting cell suspension was filtered through a nylon mesh. The adipocytes were subsequently washed three times and resuspended in DMEM containing 25 mM glucose and 10% fetal bovine serum. Incubation of primary rat adipocytes with PPARg agonists Freshly isolated mature adipocytes were incubated in 1 mL (5 105 cells/mL) of DMEM supplemented with 25 mM glucose and 10% fetal bovine serum, and treated with PPARg agonists, either rosiglitazone (10 mM) or pioglitazone (10 mM) (Enzo Life Sciences, Farmingdale, NY, USA), for 24 h at 37 C, 5% CO2. RNA isolation and expression analysis by real-time RT-PCR Total RNA was extracted from adipose tissue samples as previously described [12]. Isolated primary adipocytes after incubation with PPARg agonists were disrupted by adding 1 mL of QIAzol Lysis Reagent (Qiagen, Hilden, Germany) and homogenized for 1 min. Total RNA was then extracted with the RNeasy Lipid
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Tissue Mini Kit (Qiagen), according to the manufacturer’s instructions. The concentration and integrity of RNA samples were determined using the Experion Automated Electrophoresis System and RNA StdSens Analysis Kit (Bio-Rad, Hercules, CA, USA). mRNA expression of FSP27, PPARg2, C/EBPa, M isoform of carnitine palmitoyltransferase 1 (MCPT1), and acidic ribosomal phosphoprotein P0 (36B4) was quantified by real-time reverse transcription-polymerase chain reaction (RT-PCR) using the Chromo4 Real-Time PCR Detection System (Bio-Rad). Primers were designed with Sequence Analysis software package (Informagen, Newington, USA) from gene sequences obtained from Ensembl Genome Browser (www.ensembl.org). Primer sequences were as follows: FSP27, forward 50 -GTG TTA GCA CCG CAG ATC G-30 , reverse 50 -CAC GAT TGT GCC ATC TTC C-30 ; PPARg2, forward 50 -GAT TTG AAA GAA GCT GTG AAC C-30 , reverse 50 -GAA ATC AAC CGT GGT AAA GG-30 ; C/EBPa, forward 50 -AAG TCG GTG GAT AAG AAC AGC-30 , reverse 50 -TCA ACT CCA ACA CCT TCT GC-30 ; MCPT1, forward 50 -CAT GGT GAA CAG CAA CTA TTA CG-30 , reverse 50 -CAT CTG GTA GGA GCA CAT GG-30 ; 36B4, forward 50 GCG ACC TGG AAG TCC AAC TAC-30 , reverse 50 -TCT GCT GCA TCT GCT TGG-30 . One microgram of total RNA was converted to cDNA using the iScript cDNA Synthesis Kit (Bio-Rad). The cDNA (1% of RT reaction) was amplified by PCR using gene-specific primer pairs (0.9 mmol/L of each primer in a 20 mL reaction mixture) and iQ SYBR Green Supermix (Bio-Rad). The samples were incubated at 95 C for 5 min followed by 40 cycles of 95 C for 20 s, 55 C for 20 s, and 72 C for 40 s. Negative controls with no template cDNA (without RT) were included in each PCR. All samples were run in triplicate. SYBR Green I fluorescence was used to detect the accumulation of the PCR products and to assess their expression levels. The relative abundance of gene transcripts was determined on the basis of Ct values (Ct, threshold cycle) using the DDCt formula [17]. 36B4 was used as an endogenous reference gene to validate the Ct readings of RT-PCR and to adjust for any potential unaccounted variation or bias. Specificity of amplified PCR products was assessed by performing a melting curve analysis. PCR data were analyzed with the Opticon Monitor 3 software (Bio-Rad) and Excel (Microsoft). The results are expressed in arbitrary units, and the mean mRNA expression in control samples is set at one. Statistical analysis The results, expressed in arbitrary units, are presented as means standard error of the mean (SEM). The statistical significance of differences found between groups was assessed by one-way analysis of variance. An unpaired Student’s t test or a Kruskal-Wallis test was used to compare the groups. P < 0.05 was considered as a significant difference. Statistical analysis was performed using GraphPad Prism software (GraphPad Software, San Diego, CA, USA).
Results Effect of dietary manipulations on body weight, epididymal WAT mass, serum triacylglycerol, insulin, and leptin levels At the end of the experiment, intermittently fasted and caloric restricted rats weighed less than controls; however, changes in body weight of CR rats were less pronounced (Table 1). Moreover, dietary manipulations led to a decrease in epididymal WAT mass in rats from F, FR, IF, and CR groups when compared to the control group. The most significant reduction in epididymal WAT mass was observed in rats subjected to intermittent fasting. Similarly, serum leptin concentrations were significantly decreased in F, FR, IF, and CR as compared to control rats. Three days of food deprivation decreased circulating triacylglycerol and insulin levels in F rats relative to controls. Triacylglycerol concentrations were also lower in serum of FR and IF rats. In contrast, serum insulin levels were significantly increased after fasting-refeeding and intermittent fasting as compared to the control group. Intermittent fasting significantly up-regulates Fsp27 gene expression in rat epididymal adipose tissue Fsp27 expression was highly induced in rat WAT in response to eight cycles of food deprivation and refeeding. In IF rats, the levels of FSP27 mRNA were more than two-fold higher than in control rats (Fig. 1A). Other dietary manipulations, such as caloric restriction, fasting-refeeding, and fasting, had no effect on the expression of Fsp27 in rat epididymal adipose tissue.
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Table 1 Body weight, epididymal WAT mass, serum triacylglycerol, insulin, and leptin levels in control rats (C) and rats subjected to fasting (F), fasting and refeeding (FR), intermittent fasting (IF), and caloric restriction (CR) C Body weight [g] WAT mass [g] Serum triacylglycerols [mg/dL] Serum insulin [ng/mL] Serum leptin [ng/mL]
447.5 4.45 166.9 0.56 3.06
F
19.9a 0.34a 23.8a 0.19a 0.37a
414.9 3.28 57.0 0.09 1.63
FR
20.5a 0.39b 10.3b 0.01b 0.11b
426.2 3.48 69.8 2.14 1.86
IF
23.1a 0.40b 3.84b 0.87c 0.13b
325.9 2.37 59.21 4.55 2.13
CR
16.9b 0.14c 3.27b 0.33d 0.12b
375.9 3.72 151.3 0.86 2.05
17.2b 0.39b 10.9a 0.25a 0.20b
Values in the same row with different superscript letters are significantly different (P < 0.05)
Effect of dietary manipulations on PPARg2 mRNA levels in rat epididymal adipose tissue To elucidate whether PPARg2 is implicated in the nutritional regulation of Fsp27 gene expression in adipose tissue, PPARg2 mRNA levels were determined by real-time RT-PCR in epididymal WAT of rats subjected to intermittent fasting, caloric restriction, fasting-refeeding, and fasting. Intermittent fasting markedly up-regulated PPARg2 expression in rat WAT. In IF rats, PPARg2 mRNA levels were approximately two-fold higher than in the control group (Fig. 1B). Moreover, the expression levels of MCPT1, a known PPARg target gene in rodent white fat, were substantially increased in WAT of IF rats (Fig. 1C). In contrast to intermittent fasting, other dietary manipulations had no impact on PPARg2 mRNA expression in rat WAT. Furthermore, there was a significant positive correlation between the levels of FSP27 and PPARg2 mRNAs in adipose tissue (Fig. 2A). PPARg agonists stimulate the expression of FSP27 mRNA in isolated primary adipocytes To determine if PPARg activation might be responsible for the up-regulation of FSP27 in rat WAT, we isolated adipocytes from epididymal WAT of control rats and incubated them for 24 h with highly selective PPARg agonists, either rosiglitazone or pioglitazone. Treatment of primary rat adipocytes with PPARg agonists resulted in a significant increase in FSP27 mRNA levels (Fig. 3A). The expression of the MCPT1 encoding gene, used as a positive control for PPARg activation, was also increased in rat epididymal adipocytes treated with rosiglitazone or pioglitazone (Fig. 3B). Intermittent fasting increases C/EBPa mRNA expression in rat epididymal adipose tissue We next investigated whether C/EBPa may be involved in the nutritional regulation of Fsp27 gene expression in rat WAT. The mRNA expression levels of C/EBPa were quantified by real-time RT-PCR in epididymal adipose tissue of rats subjected to intermittent fasting, caloric restriction, fasting-refeeding, and fasting. C/EBPa mRNA levels in IF rats were markedly increased, approximately three-fold when compared to control rats (Fig. 1D). In contrast, C/EBPa mRNA expression was not altered by other dietary manipulations. Moreover, a strong positive correlation was found between Fsp27 gene expression and C/EBPa mRNA levels in rat WAT (Fig. 2B). Discussion Fsp27 was originally discovered as the adipocyte-specific gene, highly expressed only in fully differentiated adipocytes [11]. FSP27, and its human homolog, CIDEC, is expressed almost
exclusively in white adipose tissue [3,4,18]. In mice, the highest level of expression of the Fsp27 gene was found in epididymal WAT [18]. In the present work we investigated the effect of dietary manipulations, such as intermittent fasting, caloric restriction, fasting-refeeding, and fasting, on the expression of Fsp27 in rat epididymal adipose tissue. We found that intermittent fasting, in contrast to other dietary manipulations, up-regulates Fsp27 gene expression. To our knowledge, this is the first report demonstrating the induction of the Fsp27 gene transcription in response to nutritional stimuli. In cultured adipocytes, the Fsp27 gene was induced during adipogenesis [4,7,11]. The process of adipocyte differentiation is governed by two transcription factors, PPARg and C/EBPa [19]. Induced early in adipogenesis, PPARg acts as a crucial regulator of many adipocyte-specific genes and triggers the adipocyte differentiation program [19]. C/EBPa is induced relatively late during adipogenesis and is implicated in the maintenance of the adipocyte phenotype [19]. As PPARg and C/EBPa orchestrate the expression of several genes in adipocytes, we hypothesised that these transcription factors may be involved in the up-regulation of Fsp27 gene expression in WAT during intermittent fasting. PPARg2 is the isoform of PPAR restricted to adipose tissue [20]; thus, we measured PPARg2 and C/EBPa gene expression in epididymal WAT of rats subjected to intermittent fasting, caloric restriction, fasting-refeeding, and fasting. We found that mRNA levels of both transcription factors were remarkably increased in IF rats. It has been shown previously that PPARg2 gene expression in adipose tissue is not affected by diet composition [21]. However, adipose expression of PPARg2 is down-regulated by fasting and restored by refeeding [13,20]. Our studies demonstrate that PPARg2 is strongly up-regulated in WAT by multiple cycles of fasting and refeeding (intermittent fasting). Moreover, the induction of PPARg2 gene expression in white fat of IF rats was accompanied by activation of this transcription factor. In WAT of rats subjected to intermittent fasting the expression levels of MCPT1, previously identified as a PPARg target gene in rodent white fat [22], were significantly increased, indicating the enhanced transcriptional activity of PPARg. Furthermore, a significant positive correlation was found between PPARg2 and FSP27 mRNA levels in rat adipose tissue, suggesting that PPARg2 is involved in the nutritional regulation of FSP27 in WAT. Based on pharmacologic studies, one can assume that Fsp27 is a PPARg target gene. Recently, it has been demonstrated that PPARg binds directly to the PPRE element localized in the Fsp27 promoter region and increases Fsp27 promoter activity in adipocytes [9,23]. Treatment with PPARg agonist rosiglitazone increased FSP27 mRNA levels in WAT of obese and lean mice [24]. Furthermore, in vitro studies have shown that FSP27 mRNA expression in 3T3-L1 adipocytes was elevated after PPARg agonist treatment [4,23]. However, in studies published by Puri et al. rosiglitazone treatment of mature 3T3-L1 adipocytes did not influence Fsp27 gene expression [24]. These results imply that FSP27 response to
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Fig. 2. Relationship between FSP27 and PPARg2 (A) and C/EBPa (B) mRNA levels in rat epididymal WAT. Pearson’s correlation was used to determine the relationship between variables.
Fig. 1. mRNA expression levels of FSP27 (A), PPARg2 (B), MCPT1 (C), and C/EBPa (D) in epididymal WAT of control rats (C) and rats subjected to fasting (F), fasting and refeeding (FR), intermittent fasting (IF), and caloric restriction (CR). Values with different superscript letters are significantly different (P < 0.05).
PPARg agonists may be affected by the degree of differentiation of 3T3-L1 cells. Therefore, we used mature adipocytes isolated from epididymal WAT of control rats to elucidate whether PPARg may contribute to the nutritional regulation of FSP27 during intermittent fasting. In primary rat adipocytes, activation of PPARg by rosiglitazone or pioglitazone confirmed by the up-regulation of MCPT1 gene expression, led to a substantial increase in FSP27 mRNA levels, indicating that PPARg may play a significant role in the transcriptional activation of the Fsp27 gene in response to intermittent fasting. This is the first report, to our knowledge, demonstrating the induction of Fsp27 gene expression by activators of PPARg in primary adipocytes. C/EBPa was also significantly up-regulated in WAT of IF rats and its mRNA levels correlated well with the expression of Fsp27. Because interaction of C/EBPa with the Fsp27 promoter has been previously demonstrated in cultured adipocytes [11], we propose that C/EBPa is implicated in the intermittent fasting-dependent induction of Fsp27 gene expression. We found that serum insulin levels were substantially increased in rats subjected to intermittent fasting as compared to other groups. In previous studies, no association has been found, to our knowledge, between CIDEC mRNA expression in human subcutaneous adipose tissue and resting plasma insulin levels [7]. However, in vitro experiments have shown that insulin may be directly involved in the regulation of Fsp27 gene expression. In 3T3L1 adipocytes, insulin increased FSP27 mRNA levels [6]. Moreover, Ito et al. demonstrated recently that insulin was able to induce the expression of CIDEC in human preadipocytes differentiated in vitro and to increase CIDEC mRNA levels in a time- and dose-dependent manner [14]. Because insulin regulates the transcription of many adipocyte-specific genes in vivo, we assume that it is a positive regulator of Fsp27 expression during intermittent fasting.
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Fig. 3. Effect of PPARg agonists, rosiglitazone (Rosi) and pioglitazone (Pio), on FSP27 (A) and MCPT1 (B) mRNA levels in primary adipocytes isolated from epididymal WAT of control rats. Values represent means SEM of n ¼ 3 independent experiments (**P < 0.01 versus vehicle treated).
In this study, rats subjected to long-term dietary restriction, either IF or CR, gained less weight than control rats and showed reduced WAT mass, which was reflected by lower circulating leptin levels. Total calorie intake was the same between IF and CR groups; however, the reduction in WAT mass and serum triacylglycerol levels was greater in IF rats than in CR rats. This difference may result from increased fatty acid oxidation in WAT of rats subjected to intermittent fasting. Consistent with this is our finding that the gene encoding MCPT1, a key enzyme involved in b-oxidation of fatty acids in adipocytes, is up-regulated in white fat of IF rats. We have previously shown that multiple cycles of fasting and refeeding significantly increase mRNA levels and activities of lipogenic enzymes, thus enhancing lipogenesis in rat WAT [12,15,25]. The present study demonstrates that intermittent fasting may promote lipid deposition in adipose tissue by increasing the expression of FSP27, a protein involved in triacylglycerol accumulation within lipid droplets. Despite this, rats fed ad libitum after fasting-refeeding cycles remained lean and weighed less than controls [15,25]. Thus, it seems unlikely that overexpression of FSP27 in adipose tissue may contribute to the development of obesity. This is consistent with studies in humans showing that FSP27/CIDEC mRNA expression in subcutaneous adipose tissue is either uncorrelated or negatively correlated with body mass index and total fat mass [3,7]. In contrast to intermittent fasting, other dietary manipulations did not affect Fsp27 gene expression in rat adipose tissue. It is somewhat surprising that
FSP27 mRNA levels were not changed in CR rats, which received the same amount of food as IF rats, i.e., their daily food intake was reduced to 80% of control levels. These results indicate that intermittent fasting, but not a reduction in caloric intake per se, is responsible for the up-regulation of Fsp27 expression in WAT. Magnusson et al. recently reported a decrease in CIDEC mRNA levels in subcutaneous adipose tissue of morbidly obese patients undergoing treatment with a very low calorie diet [3]. However, it should be noted that these patients received less than 25% of the recommended daily caloric intake during a 16-wk treatment period. It suggests that suppression of the Fsp27 gene in adipose tissue may require a long-term and severe reduction in food intake. More modest reduction in caloric intake, as in CR rats, appears to have a minor or no effect on FSP27 mRNA levels in WAT. Taken together, these observations indicate that the regulation of Fsp27 gene expression associated with caloric restriction needs further investigation. Recent findings indicate that FSP27 is required for optimal energy storage in WAT and plays an important role in wholebody energy homeostasis. FSP27-deficient mice are protected from diet-induced obesity and insulin resistance [4,26]. However, the cellular and molecular mechanisms underlying the enhanced insulin sensitivity in FSP27-knockout mice remain largely unknown. Furthermore, the role of FSP27/CIDEC in regulating insulin sensitivity has not been fully established. In studies of obese and non-obese persons published by Keller et al., there was no association between CIDEC expression in subcutaneous adipose tissue of participants and HOMA index, or resting plasma insulin and glucose levels [7]. On the other hand, Puri et al. have found that CIDEC mRNA levels are lower in omental and subcutaneous adipose tissue of obese, insulin-resistant subjects than in insulin-sensitive subjects matched for body mass index [24]. Moreover, it has been recently reported that a loss-of-function mutation in CIDEC is associated with insulin-resistant diabetes in a female patient [10]. These latter findings suggest that reduced expression of CIDEC in adipose tissue may contribute to the pathogenesis of insulin resistance. In view of these observations, it is likely that up-regulation of FSP27 in response to intermittent fasting may enhance lipid storage in WAT, thus improving whole-body insulin sensitivity. Recent studies demonstrating the beneficial effects of intermittent energy restriction on insulin resistance in humans [27] seem to confirm this hypothesis.
Conclusion Long-term intermittent fasting, but not caloric restriction or any other dietary manipulation, significantly up-regulates Fsp27 expression in adipose tissue. Furthermore, PPARg, C/EBPa, and insulin are involved in the nutritional regulation of the Fsp27 gene in WAT. Up-regulation of FSP27 may have promoted lipid deposition in WAT after intermittent fasting; however, it did not result in the development of obesity. Further studies are needed to determine the effect of intermittent fasting-induced overexpression of FSP27 in WAT on energy homeostasis and insulin sensitivity.
Acknowledgments This work was supported by the Ministry of Science and Higher Education (2 P05A 019 30) and the Medical University of Gdansk (W-84 and ST-41).
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References [1] 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 1998;17:2526–33. [2] Liang L, Zhao M, Xu Z, Yokoyama KK, Li T. Molecular cloning and characterization of CIDE-3, a novel member of the cell-death-inducing DNA-fragmentation-factor (DFF45)-like effector family. Biochem J 2003;370:195–203. [3] Magnusson B, Gummesson A, Glad CA, Goedecke JH, Jernas M, Lystig TC, et al. Cell death-inducing DFF45-like effector C is reduced by caloric restriction and regulates adipocyte lipid metabolism. Metabolism 2008;57:1307–13. [4] Nishino N, Tamori Y, Tateya S, Kawaguchi T, Shibakusa T, Mizunoya W, et al. FSP27 contributes to efficient energy storage in murine white adipocytes by promoting the formation of unilocular lipid droplets. J Clin Invest 2008;118:2808–21. [5] Zhou Z, Toh SY, Chen Z, Guo K, Ng CP, Ponniah S, et al. Cidea-deficient mice have lean phenotype and are resistant to obesity. Nat Genet 2003;35:49–56. [6] Kim JY, Liu K, Zhou S, Tillison K, Wu Y, Smas CM. Assessment of fat-specific protein 27 in the adipocyte lineage suggests a dual role for FSP27 in adipocyte metabolism and cell death. Am J Physiol Endocrinol Metab 2008;294:E654–67. [7] Keller P, Petrie JT, De Rose P, Gerin I, Wright WS, Chiang SH, et al. Fat-specific protein 27 regulates storage of triacylglycerol. J Biol Chem 2008;283:14355–65. [8] Puri V, Konda S, Ranjit S, Aouadi M, Chawla A, Chouinard M, et al. Fatspecific protein 27, a novel lipid droplet protein that enhances triglyceride storage. J Biol Chem 2007;282:34213–8. [9] Matsusue K, Kusakabe T, Noguchi T, Takiguchi S, Suzuki T, Yamano S, et al. Hepatic steatosis in leptin-deficient mice is promoted by the PPARg target gene Fsp27. Cell Metab 2008;7:302–11. [10] Rubio-Cabezas O, Puri V, Murano I, Saudek V, Semple RK, Dash S, et al. Partial lipodystrophy and insulin resistant diabetes in a patient with a homozygous nonsense mutation in CIDEC. EMBO Mol Med 2009;1:280–7. [11] Danesch U, Hoeck W, Ringold GM. Cloning and transcriptional regulation of a novel adipocyte-specific gene, FSP27. CCAAT-enhancer-binding protein (C/EBP) and C/EBP-like proteins interact with sequences required for differentiation-dependent expression. J Biol Chem 1992;267:7185–93. [12] Karbowska J, Kochan Z, Swierczynski J. Increase of lipogenic enzyme mRNA levels in rat white adipose tissue after multiple cycles of starvationrefeeding. Metabolism 2001;50:734–8. [13] Palou M, Sanchez J, Priego T, Rodriguez AM, Pico C, Palou A. Regional differences in the expression of genes involved in lipid metabolism in adipose tissue in response to short- and medium-term fasting and refeeding. J Nutr Biochem 2010;21:23–33.
299
[14] Ito M, Nagasawa M, Hara T, Ide T, Murakami K. Differential roles of CIDEA and CIDEC in insulin-induced anti-apoptosis and lipid droplet formation in human adipocytes. J Lipid Res 2010;51:1676–84. [15] Kochan Z, Karbowska J, Swierczynski J. Unusual increase of lipogenesis in rat white adipose tissue after multiple cycles of starvation-refeeding. Metabolism 1997;46:10–7. [16] Rodbell M. Metabolism of isolated fat cells. I. Effects of hormones on glucose metabolism and lipolysis. J Biol Chem 1964;239:375–80. [17] Winer J, Jung CK, Shackel I, Williams PM. Development and validation of real-time quantitative reverse transcriptase-polymerase chain reaction for monitoring gene expression in cardiac myocytes in vitro. Anal Biochem 1999;270:41–9. [18] Puri V, Virbasius JV, Guilherme A, Czech MP. RNAi screens reveal novel metabolic regulators: RIP140, MAP4k4 and the lipid droplet associated fat specific protein (FSP) 27. Acta Physiol 2008;192:103–15. [19] Rosen ED, Hsu CH, Wang X, Sakai S, Freeman MW, Gonzalez FJ, et al. C/EBPa induces adipogenesis through PPARg: a unified pathway. Genes Dev 2002;16:22–6. [20] Escher P, Braissant O, Basu-Modak S, Michalik L, Wahli W, Desvergne B. Rat PPARs: quantitative analysis in adult rat tissues and regulation in fasting and refeeding. Endocrinology 2001;142:4195–202. [21] Rousseau V, Becker DJ, Ongemba LN, Rahier J, Henquin JC, Brichard SM. Developmental and nutritional changes of ob and PPARg2 gene expression in rat white adipose tissue. Biochem J 1997;321:451–6. [22] Wilson-Fritch L, Nicoloro S, Chouinard M, Lazar MA, Chui PC, Leszyk J, et al. Mitochondrial remodeling in adipose tissue associated with obesity and treatment with rosiglitazone. J Clin Invest 2004;114:1281–9. [23] Kim YJ, Cho SY, Yun CH, Moon YS, Lee TR, Kim SH. Transcriptional activation of Cidec by PPARg2 in adipocyte. Biochem Biophys Res Commun 2008;377:297–302. [24] Puri V, Ranjit S, Konda S, Nicoloro SM, Straubhaar J, Chawla A, et al. Cidea is associated with lipid droplets and insulin sensitivity in humans. Proc Natl Acad Sci USA 2008;105:7833–8. [25] Kochan Z, Karbowska J, Swierczynski J. The effects of weight cycling on serum leptin levels and lipogenic enzyme activities in adipose tissue. J Physiol Pharmacol 2006;57(suppl 6):115–27. [26] Toh SY, Gong J, Du G, Li JZ, Yang S, Ye J, et al. Up-regulation of mitochondrial activity and acquirement of brown adipose tissue-like property in the white adipose tissue of fsp27 deficient mice. PLoS One 2008;3:e2890. [27] Harvie MN, Pegington M, Mattson MP, Frystyk J, Dillon B, Evans G, et al. The effects of intermittent or continuous energy restriction on weight loss and metabolic disease risk markers: a randomized trial in young overweight women. Int J Obes 2011;35:714–27.