Involvement of Leptin Receptor Long Isoform (LepRb)-STAT3 Signaling Pathway in Brain Fat Mass- and Obesity-Associated (FTO) Downregulation during Energy Restriction
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Obesity is an important risk factor for cardiovascular disease, diabetes and certain cancers. The fat mass- and obesity-associated (FTO) gene is tightly associated with the pathophysiology of obesity, whereas the exact role of FTO remains poorly understood. Here, we investigated the alternations of FTO mRNA and protein expression in the peripheral metabolic tissues and the brain upon energy restriction (ER) and explored the involvement of the leptin signaling pathway in FTO regulation under ER status. ER decreased the FTO mRNA and protein expression in hypothalamus and brainstem but not in periphery. Using double-immunofluorescence staining, FTO was found to be colocalized with the leptin receptor long isoform (LepRb) in arcuate nucleus of hypothalamus and the nucleus of the solitary tract. In LepRb mutant db/db mice, the FTO downregulation in brain and body weight reduction induced by ER were completely abolished. The enhanced phosphorylation of signal transducer and activator of transcription 3 (STAT3) induced by ER was also impaired in db/db mice. Moreover, leptin directly activated the STAT3 signaling pathway and downregulated FTO in in vitro arcuate nucleus of hypothalamus cultures and in vivo wild-type mice but not db/db mice. Thus, our results provide the first evidence that the LepRb-STAT3 signaling pathway is involved in the brain FTO downregulation during ER.
Obesity has reached epidemic proportions worldwide, and accumulating evidence indicates that obesity is a heterogeneous disorder affected by both genetic and nongenetic factors. However, few genes have been identified as robust and reproducible candidates for the genetic basis of obesity so far. Recently, the association between polymorphisms of the fat mass- and obesity-associated (FTO) gene and obesity was revealed by several large human genome-wide studies (1, 2, 3). This result was subsequently confirmed by numerous investigations (4—7). Moreover, it was reported that variation in the FTO locus may also affect weight loss in humans induced by lifestyle intervention, including reduction of dietary intake (8) and moderate-intensity exercise (9). Although many studies have examined the effect of FTO gene variation on obesity risk, the biological functions of FTO in obesity and the underlying mechanisms are largely unknown.
In rodents, it was reported that the FTO mRNA is widely expressed in many tissues, with especially high expression in the hypothalamus and pancreatic islets (10). Some reports have indicated that the FTO gene seems to exhibit its function in the central nervous system (CNS) by modulating energy homeostasis (10, 11, 12). Hypothalamic FTO mRNA expression was downregulated by fasting in mice (10,11) but was upregulated in rats (12). However, the exact influence of long-term energy restriction (ER) on FTO protein levels has never been determined. Furthermore, apart from the CNS mechanism, whether FTO in peripheral metabolic tissues is involved in the energy intake/expenditure and obesity is unclear.
It has been well established that leptin plays a critical role in feeding and energy storage (13, 14, 15). Leptin regulates energy balance throughout the body by controlling processes involved in energy intake and utilization both in the periphery and the brain (13,16). Leptin acts through its receptor to regulate the downstream signaling pathways. Alternative splicing of the transcript from a single leptin receptor gene produces multiple leptin receptor isoforms, of which the long isoform (LepRb) is the major signaling form of the leptin receptor (17), especially in brain (13). ER is a common and robust intervention used to reduce body weight in obese patients. Moreover, it was reported that ER can enhance leptin sensitivity by enhancing both LepRb expression and signaling capacity in the brain to prevent central leptin resistance (18,19). Given that ER is one of most effective methods for decreasing body weight and FTO is thought to be involved in the pathophysiological process of obesity, we therefore consider the clarification of the association of the leptin signaling pathway and FTO valuable. Although serum leptin levels were reported to be associated with variants of the FTO gene (20, 21, 22), the association between FTO and the leptin signaling pathway is largely unknown. Thus, we tested whether ER for 8 or 50 weeks can alter FTO expression in the periphery and CNS. After finding the modulation of FTO was limited in the brain, we hypothesized that the CNS LepRb may be of important physiological significance in modulating FTO expression upon ER.
Materials and Methods
Animals and Reagents
The animal studies were performed in accordance with the Guide for Care and Use of Laboratory Animals published by the National Academy Press (1996). Eight-week-old male Sprague-Dawley rats were purchased from Sino-British SIPPR/BK Lab Animal Ltd. (Shanghai, China). Ten-week-old male BKS.Cg-Dock7m +/+ Leprdb/J mice (BKS-db/db, The Jackson Laboratory stock number 000642) and their lean C57BLKS/J wild-type (WT, The Jackson Laboratory stock number 000662) controls were obtained from the National Model Animal Research Center (Nanjing, China). Animals were housed in controlled conditions (temperature 23 ± 2°C, humidity 60 ± 10%, and lighting from 8:00 AM to 8:00 PM), with free access to water.
FTO polyclonal antibody, LepRb goat polyclonal antibody (SC-1832, recommended for detection of long form of leptin receptor long isoform [LepRb]) and signal transducer and activator of transcription 3 (STAT3) inhibitor S3I-201 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Another LepRb chicken polyclonal affinity purified antibody (catalog number CH14104) was from Neuromics (Edina, MN, USA). Recombinant leptin was purchased from Phoenix Pharmaceuticals Inc. (Phoenix, AZ, USA). The FTO (clone number H-300, SC-98768) is a rabbit polyclonal antibody raised against amino acids 252–505 mapping at the C-terminus of FTO of human origin. Because these amino acids were identical among human, rat and mouse, this antibody could be used to detect FTO protein from the three species. The specificity of this antibody was tested in our lab (data not shown) and by the manufacturer (https://doi.org/www.scbt.com/picts.php?pict_id = 162218). Anti-phospho-STAT3Tyr705, anti-total-STAT3, anti-phospho-Jak2Tyr1007/1008 and anti-total-JAK2 were purchased from Cell Signaling Technology (Beverly, MA, USA). Anti-phosphoSer9-GSK3, anti-total-GSK3 and JAK2 inhibitor AG-490 were purchased from Calbiochem (San Diego, CA, USA). Anti-β-actin and GSK-3 inhibitor L803-mts were purchased from Sigma (St. Louis, MO, USA). IRDye 700CW- or 800CW-conjugated secondary antibodies were obtained from Li-Cor Bioscience (Lincoln, NE, USA). Dylight 488-conjugated goat antirabbit and Cy3-conjugated goat antimouse IgG were purchased from Jackson ImmunoResearch Inc. (West Grove, CA, USA).
After 1 wk of acclimation, rats were divided into two groups. The animals in the energy restriction group (ER group) had access to 60% of the ad libitum consumption of the control animals (ad libitum [AL] group). The standard chow for rats or mice was purchased from Sino-British SIPPR/BK Lab Animal Ltd. This chow diet contained a variety of nutrient substances including 20% protein, 5% fat and other essential vitamins/microelements. The amount of food intake of AL animals was measured every week to adjust the amount of ER animal food intake weekly. Body weights of rats were recorded every week. After 8 and 50 wks of ER, rats were anesthetized with pento-barbital (40 mg/kg) and blood was swiftly collected. The mesenteric adipose was considered to be visceral adipose. Hypothalamus and brainstem were dissected according to the method described by Glowinski and Iversen (23) and Andrews and Murphy (24). Tissues were harvested as described previously (25). Serum glucose levels were measured as described previously (26). Serum insulin levels were determined by an enzyme-linked immunosorbent assay kit (Linco Research, St. Louis, MO, USA).
After 1 wk of accommodation, WT and db/db mice were divided into four groups: WT-AL, WT-ER, db/db-AL and db/db-ER groups. Given that the food intakes of WT mice were not the same as those of db/db mice, WT-ER mice were subjected to ER with 60% of food compared with WT-AL mice, and db/db-ER mice were subjected to ER with 60% of food compared with db/db-AL mice, respectively. Mice body weights were recorded every week. After 8 wks of ER, mice were anesthetized with pentobarbital (40 mg/kg), and the hypothalamus and brainstem were harvested.
Reverse Transcript Polymerase Chain Reaction and Real-Time Polymerase Chain Reaction
Total RNA was extracted from tissues using Trizol (Invitrogen, Carlsbad, CA, USA) as described previously (27), and 1 μg RNA was reverse-transcribed to cDNA using RNA reverse transcriptase (Promega, Madison, WI, USA) (28). Polymerase chain reaction (PCR) products were electrophoretically separated with 1.2% agarose gels. Real-time PCR was performed using an Opticon Monitor 3 Real-Time PCR System (Bio-Rad, Hercules, CA, USA) and SYBR Premix ExTaq Mixture (Takara, Otsu, Japan) as described previously (29). The following primers were used: FTO (for real-time PCR), forward: 5’-GCCGCATGTC AGACCTTCC-3’, reverse: 5’-GACCT GTCCACCAAGTTCTCG-3’; FTO (for reverse transcript [RT]-PCR), forward: 5’-TTTGCACTGATTGGTGGTGTG-3’, reverse: 5’-GGACGGCAGACAGAA TTTCACT-3’; β-actin, forward: 5’-AGACC TCTATGCCAACACAGTGC-3’, reverse: 5’-GAGC CACCAATCCACACAGAGT-3’. β-Actin was used as internal control, and the amount of RNA was calculated by the comparative threshold cycle method as recommended by the manufacturer. The real-time PCRs were performed with transcription efficiency >90%. All the data were calculated from duplicate reactions.
Western blotting analysis was performed as described in our previous reports (30). Tissues were homogenized by protein extraction reagent (Beyotime, Nanjing, China) supplemented with protease inhibitor cocktail (Pierce, Rockford, IL, USA) and phosphatase inhibitor cocktail (Pierce). About 20 of samples were run on 10% SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis). The proteins were electrotransferred to nitrocellulose membranes. The nitrocellulose membranes were incubated with primary antibody for 2–3 h at 25°C and then incubated with corresponding IRdye-800CW- or 700CW-conjugated secondary antibodies. The images were captured and analyzed by the Odyssey infrared fluorescence imaging system (Li-Cor Bioscience). The adoption of this system allowed for superior sensitivity, linearity and reproducible quantitation compared with formerly used X-ray film-based technologies for Western blotting (31). Each experiment was repeated at least three times.
Immunofluorescence analysis was performed as described previously with some modification (29). Frozen 20-[μm-thick rat brain sections were fixed in 4% paraformaldehyde, blocked by 8% normal goat serum and incubated in specific primary antibodies (FTO, rabbit polyclonal antibody, 1:100 dilution; LepRb, mouse monoclonal antibody, 1:100 dilution) at 4°C overnight. After being washed three times with phosphate-buffered saline, the sections were incubated with Dylight 488-conjugated goat antirabbit or Cy3-conjugated goat antimouse IgG. Normal goat IgG was used for the negative control. No visual fluorescence was observed in the negative control. The images were recorded using a confocal laser scanning microscope (Olympus, FV1000, Tokyo, Japan) and analyzed using Image Pro Plus software.
Isolated Arcuate Nucleus of Hypothalamus Explant Cultures
Isolated arcuate nucleus of hypothalamus (ARH) explant cultures were prepared according to a previous report with some modifications (32). The hypothalamuses were collected under anesthesia (sodium pentobarbital, 40 mg/kg i.p.) from postnatal day 7 WT mice and then sectioned at 200~300 [μm. The ARH was carefully dissected under a stereo-microscope. Explants were cultured onto poly-L-lysine precoated glass coverslips and incubated in serum-free medium (33). The EOL-1 medium containing either recombinant leptin (100 ng/mL) or vehicle was replaced every day. After 1 or 3 d in vitro, the explants were washed with ice-cold phosphate-buffered saline and then lysed for Western blotting analysis.
Intracerebroventricular Injection of Leptin
Intracerebroventricular injection of leptin was performed as described previously with some modifications (34). The coordinates for lateral ventricular injection were as follows: anterior-posterior (from bregma): -0.5 mm; dorsal-ventral (from skull surface): -2.5 mm; and medial-lateral: 1 mm. A single-bolus leptin (1 μg, 1 μg/μL) was injected into the lateral ventricle. Four hours later, rats were killed and the hypothalamuses were harvested for immunoblotting.
Data are represented as means ± SD. Differences were evaluated by two-tailed Student t test (two groups) or one-way analysis of variance followed by Tukey post hoc test (three or more groups). Statistical significance was set at P < 0.05.
FTO mRNA Expression in Tissues
ER Downregulates FTO mRNA Expression in Brain Rather Than in Peripheral Tissues
Characteristics of AL and ER rats.
AL 8 wks (n = 8)
ER 8 wks (n = 8)
AL 50 wks (n = 4)
ER 50 wks (n = 4)
Body weight (g)
455 ± 11
325 ± 4a
535 ± 32
412 ± 26b
Left anterior gastrocnemius muscle (g)
1.08 ± 0.04
1.00 ± 0.03
1.42 ± 0.10
1.02 ± 0.04b
15.99 ± 0.72
9.53 ± 0.22a
11.87 ± 0.72
9.37 ± 0.22c
Subcutaneous fat (g)
18.65 ± 0.73
10.16 ± 0.60a
20.32 ± 2.19
9.57 ± 0.89c
Visceral fat (g)
19.35 ± 1.20
6.85 ± 0.18a
21.90 ± 2.12
14.55 ± 0.79b
Epididymal fat (g)
6.12 ± 0.44
2.59 ± 0.15a
7.59 ± 0.70
5.67 ± 0.44b
Retroperitoneal fat (g)
6.55 ± 0.68
1.59 ± 0.20a
8.32 ± 1.18
4.34 ± 0.36b
Perirenal fat (g)
2.36 ± 0.20
0.75 ± 0.05a
1.81 ± 0.04
1.68 ± 0.16
Mesenteric fat (g)
4.32 ± 0.37
1.92 ± 0.08a
4.18 ± 0.35
2.85 ± 0.05c
Serum glucose (mmol/L)
7.26 ± 0.45
8.08 ± 0.33
7.48 ± 0.28
7.65 ± 0.60
Serum insulin (mmol/L)
0.55 ± 0.05
0.26 ± 0.06a
0.47 ± 0.04
0.29 ± 0.02b
FTO Protein Expression in Brain Is Downregulated by ER
Colocalization of FTO and LepRb in Hypothalamus and Brainstem
Reduction of Body Weight and Downregulation of Brain FTO Induced by ER Are Impaired in db/db Mice
Activation of STAT3 Signaling by ER in Brain Is Impaired in db/db Mice
Leptin Downregulates FTO in Isolated ARH Cultures via STAT3 but Not the GSK-3 or JAK2 Signaling Pathway
Leptin Downregulates FTO and Activates the STAT3 Signaling Pathway In Vivo
A single bolus leptin injection into lateral ventricle decreased hypothalamic FTO expression in WT mice but not in db/db mice (Figure 6C). Intracerebroventricular injection of leptin also activated phosphorylation of STAT3 in WT mice but not in db/db mice (Figure 6D). Moreover, we observed a significant increase of phosphorylation of STAT3 in WT-ER mice compared with WT-AL mice (see Figure 6D). Interestingly, leptin further increased p-STAT3 levels in WT-ER mice (see Figure 6D). Meanwhile, FTO was downregulated by ER and leptin (see Figure 6D). However, all these changes were not observed in db/db mice (see Figure 6D).
Emerging evidence suggests that the FTO gene is an obesity gene controlling food intake or energy expenditure, or both (1,2). In the present study, we demonstrated that ER was able to decrease both mRNA and protein expression of FTO in brain rather than in peripheral metabolic-sensitive tissues. Moreover, we found that the LepRb was essential for the effect of ER on FTO expression, and the STAT3 signaling pathway contributed to this process.
As far as we know, this is the first investigation addressing the FTO protein expression pattern in tissues and how FTO protein changes after ER. Consistent with several previous reports of FTO mRNA (10,12,35), we detected high mRNA expression of FTO in hypothalamus and brainstem. Additionally, we confirmed the high protein expression of FTO in hippocampus and brainstem.
This finding indicates that the pattern of FTO protein expression is in accordance with the FTO mRNA expression pattern in brain. Furthermore, we found that the expression of the FTO gene in skeletal muscle and liver was higher than that in adipose tissue. Some researchers focused on adipose tissue to explore the relationship between the FTO gene and obesity. A previous report showed that FTO mRNA expression in subcutaneous adipose tissue is higher than in visceral adipose tissue (36), whereas another publication reported the reverse results (11). We showed that FTO mRNA expression in visceral adipose tissue was two- to three-fold higher than that in subcutaneous adipose tissue, whereas the FTO protein expressions were comparable in these two fat pads.
It should be noted that how the expression of FTO changes upon ER is still disputable. Previous reports showed that FTO mRNA was downregulated in mouse hypothalamus (10,11) but upregulated in rat hypothalamus (12) after food deprivation (that is, fasting for 1–2 days). We considered it might be more reasonable that the rat and mouse share the same or similar regulating pattern of FTO expression. In this study, we found the decline of FTO mRNA and protein expression in hypothalamus and brain-stem both in ER rats and mice. These results support the results of Gerken et al. (10) and Stratigopoulos et al. (11) in mice. However, the results may partly conflict with observations showing that FTO is upregulated during food deprivation in rats to some extent (12). However, the difference in treatment between our study and the above-mentioned studies should be emphasized. This study was designed to investigate the FTO expression change in calorically restricted animals for ≥8 weeks, whereas the previous studies focused on the influence of short-term food deprivation for 1–2 days on FTO expression. These differences may account for the different results.
Furthermore, we did not find any change of FTO mRNA or protein expressions in the peripheral metabolic-sensitive tissues in ER rats. These results suggest that the FTO gene in the peripheral metabolic tissues does not directly regulate metabolic processes under ER. Considering the high FTO expression in skeletal muscle and liver and that the FTO gene encodes a 2-oxoglutarate-dependent demethylase in the nuclei (10), even though we failed to find any change of FTO gene expression in peripheral metabolic tissues of ER rats, these results do not rule out that FTO may play a role in regulating biological function via posttranslational mechanisms in liver and muscle.
The most important finding of our study is the LepRb-dependent regulation of FTO expression during ER. It has been well established that hypothalamus and brainstem play key roles in generating homeostatic responses to regulate food intake and energy expenditure, and leptin is one of the most important hormones controlling the feeding and energy balance in these two brain areas (13,16,37). Moreover, several intracellular signaling pathways, such as STAT3, JAK2 and GSK3, were reported to contribute critically in the effects of leptin on neuropeptide production and body energy homeostasis (38, 39, 40, 41, 42, 43). In our study, we found that the FTO colocalizes with LepRb in hypothalamic and brainstem neurons. Thus, FTO might be regulated by leptin via LepRb-mediated intracellular signaling. To confirm this speculation, we tested whether ER can modulate FTO expression in LepRb-mutated db/db mice, a widely used obese animal model (16). In normal status, there is no difference of FTO expression between WT and db/db mice. FTO expression in hypothalamus and brainstem decreased significantly after 8 wks of ER in WT mice but not in db/db mice, indicating LepRb mutation impaired the effects of ER on FTO expression. We also found that the exogenous leptin treatment reduced FTO expression in in vitro ARH cultures. Moreover, stereotaxically intracerebro-ventricular microinjection of leptin indeed directly decreased the brain FTO expression in WT mice but not in db/db mice, indicating the brain FTO down-regulation is LepRb dependent.
We noted that the circulating leptin level was lower (about 0.3–0.4-fold) in ER than in normal conditions (18,44). However, the LepRb expression in brain under ER conditions was increased significantly (18,19). The response to leptin under ER conditions is even more sensitive than under normal conditions. In fact, the ER rats or mice exhibited an increased response to leptin in brain (15,18,19,45). This enhanced leptin sensitivity under ER conditions was able to counteract the leptin resistance caused by aging and diet-induced obesity (15,18,19,45). Recently, Church et al. (46) demonstrated that circulating leptin levels were lower in FTO transgenic mice. Additionally, Stratigopoulos et al. (47) showed that FTO regulated LepRb trafficking to the cilium and subsequently modulated cellular response to leptin. Thus, the relationship between FTO and leptin/LepRb may be complex and needs to be further investigated.
Among the LepRb-related signal pathways, we demonstrated that only the STAT3 activation induced by ER was abolished in db/db mice (see Figure 5), whereas the effects of ER on JAK2 and GSK3 pathways did not change in db/db mice. Leptin treatment directly down-regulated FTO in ARH cultures in vitro, which could be abolished by the STAT3-specific inhibitor but not the GSK-3 or JAK2 inhibitor (Figure 6B). Moreover, leptin injection reduced FTO expression (Figure 6C) and activated the STAT3 signaling pathway (Figure 6D) in WT but not in db/db mice in vivo. Therefore, the change trends of FTO and p-STAT3 make us believe that the LepRb-STAT3 signaling pathway is critically involved in the downregulation of brain FTO during ER. Tung et al. (48) demonstrated that overexpression of FTO in rat ARH using adeno-associated virus not only decreased food intake, but also resulted in a four-fold increase in the mRNA levels of STAT3. They also considered that the STAT3 might be a possible candidate for the mediation of FTO actions.
In conclusion, we have demonstrated that ER can decrease FTO mRNA and protein expression in both rat and mouse brains. The LepRb-STAT3 signaling pathway is involved in this process, not only evidenced by the absence of FTO down-regulation and impairment of STAT3 activation in food-restricted db/db mice, but also evidenced by exogenous leptin activating the STAT3 signaling pathway and decreasing FTO expression in vitro and in vivo. Therefore, these findings suggest an involvement of the LepRb-STAT3 signaling pathway in brain FTO downregulation by ER.
The authors declare that they have no competing interests as defined by Molecular Medicine, or other interests that might be perceived to influence the results and discussion reported in this paper.
This work was supported by grants from the National Natural Science Foundation of China for Distinguished Young Scholars to C-Y Miao (30525045), the National Basic Research Program of China to C-Y Miao (2009CB521902), the National Science and Technology Major Project to C-Y Miao (2009ZX09303-002), the Program of Shanghai Subject Chief Scientist to C-Y Miao (10XD1405300), the Open Funds of the Shanghai Key Laboratory of Vascular Biology to P Wang (GXY2009001005) and the “Shizhen Project” of the School of Pharmacy, Second Military Medical University, China, to PWang.
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