Contribution of irisin pathway in protective effects of mandarin juice (Citrus reticulata Blanco) on metabolic syndrome in rats fed with high fat diet


 The beneficial effects of Citrus fruits and their secondary metabolites on the cardiovascular system are well established. Moreover, growing evidence suggests beneficial role for prevention of obesity and related dysfunctions. Citrus reticulata Blanco (Rutaceae) is one of the most consumed Citrus fruits, but it is poorly investigated. Mandarin juice obtained from C. reticulata fruits, collected in the Horti Simplicium of Pisa Charterhouse, presents a high amount of flavanone glycosides, including hesperidin and a number of polymethoxyflavonoids, in particular nobiletin and tangeretin. On Wistar rats fed with a high fat diet for 21 days, mandarin juice significantly contained the percentage weight gain, reduced visceral adipose tissue and the inflammatory markers TNF and IL-6. Furthermore, mandarin juice influenced irisin pathway, increasing its plasma levels. Finally, supplementation with mandarin juice contributed to improve mitochondrial membrane potential, partially compromised with high fat diet, making mitochondria less susceptible to harmful events, such as ischemia. Taken together, these results suggest that C. reticulata, through its main metabolites, is able to produce beneficial effects in metabolic syndrome and to promote browning process, through involvement of the novel interesting irisin pathway.



| INTRODUCTION
Metabolic syndrome (MS) is a condition affecting at least 30% of adults in the Western society, but it is widespread also in the urban population of some developing countries (Saklayen, 2018). It is characterized by the presence of, at least, three following variables: elevated waist circumference, high levels of blood pressure, insulin resistance, and dyslipidemia (Sirtori, Pavanello, Calabresi, & Ruscica, 2017). MS is strongly associated with increased risk of developing diabetes and atherosclerotic and nonatherosclerotic cardiovascular diseases (Mottillo et al., 2010). Very recent studies highlighted a close correlation between MS and serum irisin levels: indeed, lower levels of this adipomyokine were found in the serum of patients with MS, as well as type 2 Diabetes Mellitus. Noteworthy, irisin is involved in white adipose tissue browning and in energy expenditure, insulin sensitivity, and antiinflammatory pathways, therefore it is emerging as a crucial player in metabolic disorders and as a possible target to mitigate these conditions (Huerta-Delgado et al., 2020). Lara Testai and Marinella De Leo equally contributed to the work.
As concern the active metabolites, the Citrus flavonoids, such as naringenin and nobiletin, have been associated with preventive effects against the development of metabolic disorders and atherosclerosis (Alam et al., 2014;Burke et al., 2018). Recently, another Citrus flavanone, hesperidin, has been identified as a regulator of both lipid and glucose metabolism through the activation of AMPK-PPAR pathway (Xiong et al., 2019). These results lead to hypothesize that such beneficial effects can be attributed to active metabolites and therefore can be considered as property of other Citrus fruits. In particular, Citrus reticulata

Blanco (Rutaceae) is one of the most widely consumed and distributed
Citrus fruits all over the world (Wang et al., 2017). However, the knowledge of its cardiovascular profile is lacking. Previous investigation about the chemical content of different varieties of C. reticulata showed that, similarly to other Citrus fruits, flavedo, albedo, and juice are rich in flavonoids, such as flavones, flavanones, and polymethoxyflavones (Russo, Cacciola, Bonaccorsi, Dugo, & Mondello, 2011;Wang et al., 2017;Zhang et al., 2014), thus arousing a great interest in their potential biological value. The investigation on C. reticulata fruits, performed in this study, is combined to the valorization of an Italian cultural heritage, since the plant species was collected in the Horti Simplicium of Pisa Charterhouse, an ancient monastery located in Calci (Pisa, Italy). After the abandon of last monks at the beginning of the 1970s, it became a National Museum and hosted later the Natural History Museum of the University of Pisa. Therefore, in the context of the requalification of Pisa Charterhouse Citrus plants, the juice of C. reticulata has been evaluated for its in vivo protective effects on metabolic syndrome and its content in terms of bioactive substances.

| Chemicals
HPLC grade acetonitrile and formic acid were purchased from VWR (Italy). HPLC grade water (18 MΩ) was prepared by using a Mill-Ω 50 purification system (Millipore Corp., MA, USA). N,N-dimethylformamide (DMF) was purchased from Alfa Aesar (Germany). Naringin and vitexin 2 00 -O-glucoside were isolated in our laboratory from plant materials during previous studies and fully characterized by 1D-and 2D-NMR analyses.

| Chemical characterization of mandarin juice by HPLC-PDA/UV-ESI-MS/MS
Defrosted juice (10 mL) was mixed to an equal volume of DMF, centrifuged three times for 5 min at 1,145×g, then the supernatant was filtered through PTFE membrane (3 mm, 0.45 μm) before HPLC analyses.
The LC-photodiode array (PDA)-mass spectrometry (MS) system was composed by a Surveyor autosampler and a Surveyor LC pump, interfaced with a Surveyor PDA/UVvis detector and a LCQ Advantage ion trap mass spectrometer equipped with an electrospray ionization (ESI) source (ThermoFinnigan, CA, USA). A Synergi Fusion-RP column (4.6 × 250 mm, 4 μm, Phenomenex, Italy) was used for chromatography, eluting with a mixture of acetonitrile (solvent A) and water (solvent B) using the solvent gradient reported by Da Pozzo et al. (2018). Elution (20 μL injection volume) was performed at 0.8 mL/min flow rate, with a splitting system of 2:8 to MS detector (160 μL/min) and PDA detector (640 μL/min), respectively. PDA/UV spectra were acquired in a wave-

| Animals
In vivo experiments were carried out according to European (EEC Directive 2010/63) and Italian (D.L. March 4, 2014 n.26) legislation (491/2018-PR); moreover, ARRIVE guidelines have been put into practice. Animals were housed in cages with food and water ad libitum, and they were exposed to a 12 h dark/light cycle. The experiments were conducted on male Wistar rats (ENVIGO) with a body weight of 336 ± 8 g.

| Chronic in vivo treatment
The animals were randomly divided into three groups (6 animals per group) and treated for 21 days: one group was treated with a standard diet (STD, ENVIGO; for composition Table 1). The second one was treated with a high fat diet (HFD, SAFE; for composition see Table 1). The third one was treated with HFD + mandarin juice 24% v/v (HFD + MJ) diluted in water. The dosage of mandarin juice has been established on the basis of previous epidemiological and clinical studies in which other Citrus juices have been used (Morand et al., 2011;Testai & Calderone, 2017). The water intake was evaluated daily, instead the body weight and the food intake three times a week for each animal.
At the end of the treatment each animal was deprived of food and after 24 h was anesthetized with Thiopental Sodium (70 mg/ kg, MSD animal health). The blood was collected, through the caudal vein, to perform blood glucose test (Glucocard G meter, Menarini Diagnostics). Subsequently the animals were sacrificed with an overdose of Thiopental Sodium. Blood was collected in tubes with the anticoagulant EDTA (BD Vacutaine) by intracardiac sampling and organs (heart, liver, and abdominal adipose tissue) were removed, weighed, and stored for functional and enzymatic investigations.

| Evaluation of blood lipid panel
Blood was immediately used for the evaluation of lipid panel (total cholesterol, HDL, LDL, triglycerides) and glycated hemoglobin levels using Cobas b101 instrument (Roche Diagnostics, Milan, Italy). Residue samples were centrifuged at 1,000×g for 10 min, obtaining plasma, which was frozen at −20 C for subsequent analysis.
Data analysis. Blood samples were collected from six animals per group and results were expressed as mean ± SEM. Cardiovascular risk was calculated as ratio between total cholesterol and HDL cholesterol levels. One-way ANOVA followed by Bonferroni's post hoc test was used to compare groups for statistical differences (p < .05).
2.7 | Functional analysis of cardiac mitochondrial membrane potential 2.7.1 | Cardiac mitochondria isolation The heart was cut into small 2/3 mm 3 pieces in STE (composition: sucrose 250 mM, Tris 5 mM, EGTA 1 mM; pH 7.4) and constantly on ice (4 C). The pieces were transferred in STE (4 C) and then homogenized with Ultra-Turrax (model: IKA, T-18 Basic). After three homogenization cycles of about 20 s, the first centrifugation was carried out: 1,090×g, 3 min, 4 C. The supernatant was transferred to a new tube kept on ice and the pellet was resuspended in STE. The suspension obtained was centrifuged again under the same conditions. Then, the supernatants were picked up and centrifuged again but under different conditions: 11,970×g, 10 min, 4 C. Two pellets were obtained and the supernatants were discarded. Each of them was resuspended in STE and centrifuged again at 11,970×g for 10 min, 4 C. The pellet was preserved and resuspended in ST (composition: sucrose 250 mM, Tris 5 mM; pH 7.4) and centrifuged using the previous conditions. The pellet obtained, corresponding to the mitochondrial fraction, was resuspended in 400 μL of ST and transferred to a frozen eppendorf.
The mitochondrial protein concentration in the supernatant was determined spectrophotometrically by Bradford assay (Bio-Rad, USA), using a microplate reader (EnSpire, PerkinElmer, USA).
where ΔΨm is the mitochondrial membrane potential (mV), V 0 is the volume of the incubation medium before the addition of mitochondria, V t is the volume of the incubation medium after the addition of the mitochondria, V m is the volume of the mitochondrial matrix (taken as 1 μL/mg protein), [TPP + ] 0 and [TPP + ] t represent, respectively, the TPP + concentrations recorded before addition and at time t, P is the protein concentration expressed in mg, K 0 and K i are apparent external and internal partition coefficients of TPP + (14.3 and 7.9 μL/mg, respectively). The mitochondrial membrane potential was evaluated on six different animals per group, and data were expressed as mean ± SEM. One-way ANOVA followed by Bonferroni's post hoc test was used to compare groups for statistical differences (p < .05).

| Evaluation of citrate synthase activity on adipose tissue
Frozen adipose tissues were homogenized on ice with an ultra-turrax homogenizer (IKA-Werke GmbH & Co., Germany) in a cold buffer Data analysis. The assay was performed on six animals per group.
Citrate synthase activity was expressed in mU/μg protein. Data were analyzed by a computer fitting procedure (software: GraphPad Prism 5.0). Results were expressed as mean ± SEM. One-way ANOVA followed by Bonferroni's post hoc test was used to compare groups for statistical differences (p < .05).

| Evaluation of inflammatory markers
The levels of TNF and IL-6 were evaluated in adipose tissues using ELISA commercial kits (AbFRONTIER, Korea). About 500 mg of adipose tissue have been thawed, added with 500 μL of STE (pH 7.4, 4 C), and homogenized with potter. The suspension was transferred to an eppendorf and centrifuged for 15 min, at 12,000×g and 4 C. The supernatant was used to make the appropriate dilutions.
Data analysis. One-way ANOVA followed by Bonferroni's post hoc test was selected as statistical analysis, and the difference between groups was considered statistically different when p < .05.

| Investigation of irisin pathway
Irisin's levels were measured directly in plasma using an ELISA commercial kit (Adipogen Life Science, Switzerland). For subsequent passages of the ELISA kits, the protocols indicated by the manufacturer were followed.
Moreover, the downstream mediators were evaluated in adipose tissue (about 100 mg) by using the RNeasy Mini Kit (Qiagen, Germany), according to the manufacturer's protocol.
Then, total RNA was extracted and converted into first-strand cDNA by using the QuantiNova Reverse Transcription Kit (Qiagen, Germany  According with the literature, rats fed for 21 days with HFD showed significantly higher levels of blood glucose, total cholesterol, triglycerides, and LDL if compared with STD diet (Table 2). Moreover, HDL value was markedly reduced (Sullivan, Cerda, Robbins, Burgin, & Beatty, 1993). MJ did not improve HDL, but clearly reduced blood glucose, total cholesterol, triglycerides, and LDL levels, significantly containing cardiovascular risk (Table 2). This improvement of lipid profile has been also described for other Citrus fruits, among which ber- Interestingly, in this experimental model, in adipose tissue of rats fed with HFD, higher levels of pro-inflammatory cytokines, IL-6 and TNF were observed (Figure 2a,b). Conversely, CS activity was compromised. Oral supplementation with MJ clearly contributed to reduce cytokine release and improve the CS activity, suggesting that such a nutraceutical intervention could ameliorate metabolism and contain inflammation (Figure 2c).
Growing evidence points out the key role of irisin in several metabolic disorders, including obesity (Flori, Testai, & Calderone, 2021).
Irisin is a newly characterized myokine that promotes energy expenditure by white adipose tissue browning; indeed, irisin, secreted from skeletal muscle to blood, is responsible, through the stimulation of UCP-1, for thermogenesis, a well-known mechanism to loss body weight. Therefore, this discovery has generated great interest, being a F I G U R E 1 Effects of mandarin juice on body weight and tissue mass.  . Indeed, PGC-1α is a master regulator of mitochondrial biogenesis, that contributes to increase mitochondrial ATP synthesis and oxidative metabolism, such as oxidative phosphorylation and fatty acid β-oxidation; its levels are reduced in HF diet if compared to low fat diet (Kazeminasab et al., 2018). Finally, peroxisome proliferator-activated receptor γ (PPARγ) has been also found to promote mitochondrial biogenesis in adipocytes, accompanied with increased thermogenesis and browning (Liu, Wang, & Lin, 2019 Figure 3).
Accordingly, lower levels of irisin have been measured in the blood of HFD-treated animals. In contrast, irisin levels in HFD + MJ group were almost equivalent to that recorded in STD group ( Figure 3d). Moreover, the supplementation with MJ counteracted in a significant manner the HFD-induced decrease in the transcriptional expression levels of PPARG and PPARGC1A genes (Figure 4a,b), coding, respectively for PPARγ and PGC-1α.
In addition to the decreased expression of PPARγ and PGC-1α and the circulating irisin levels, HF diet induced also a significant decrease of transcriptional levels of UCP-1 (Figure 4c). This decrease was almost absent in the adipose tissue from MJ-treated rats, confirming the role of irisin pathway as a target in the effects of MJ, perfectly in agreement with the increase in UCP-1 gene expression and UCP-1 protein abundance observed in the classical brown adipose tissue depots (Zhang et al., 2016).
Therefore, such results suggest that the nutraceutical supplementation with mandarin juice can stimulate the browning mechanism on adipose tissue through the stimulation of irisin pathway.
Considering that cardiovascular diseases are the main consequences of metabolic syndrome and obesity (Galassi, Reynolds, & Note: * indicates a significant statistical difference between HF and STD groups. § indicates significant statistical difference between HF and HF + MJ The single symbol corresponds to p < .05; the double symbol to p < .01, and the triple symbol to p < .001. F I G U R E 2 Effects of mandarin juice on pro-inflammatory cytokines and cell metabolism. (a) Change of IL-6 levels in adipose tissue.
(b) Change of TNF levels in adipose tissue. (c) Activity of the Citrate Synthase enzyme in adipose tissue. * indicates a significant statistical difference between HF and STD groups. § indicates significant statistical difference between HF and HF + MJ. The single symbol corresponds to p < .05; the double symbol to p < .01, and the triple symbol to p < .001 He, 2006), the cardiac mitochondrial membrane potential was evaluated as an indicator of cardiomyocytes tolerance toward possible harmful events. Indeed, modest, but significant, depolarization of mitochondrial membrane has been recognized in hearts of HFD-fed animals and it has been associated with a lower bioenergetic efficacy.
This makes mitochondria more susceptible to ischemic events. Interestingly, cardiac mitochondria from rats fed with HFD + MJ showed an inner membrane potential almost equal to organelles of STD group ( Figure 5).

| Chemical profile of mandarin juice
The chemical investigation on mandarin juice was carried out by means of HPLC-PDA/UVvis-MS/MS techniques. In total, 14 flavonoids were tentatively identified comparing their elution order, UV and ESI-MS data (Table 3) with those reported in previous studies (Russo et al., 2011;Wang et al., 2017;Zhang et al., 2014). * indicates a significant statistical difference compared to HF group. The single symbol corresponds to p < .05, the double symbol to p < .01, and the triple symbol to p < .001. * indicates a significant statistical difference between HF and STD groups. § indicates significant statistical difference between HF and HF + MJ. The single symbol corresponds to p < .05; the double symbol to p < .01, and the triple symbol to p < .001 F I G U R E 5 Effects of mandarin juice on cardiac mitochondrial membrane potential. * indicates a significant statistical difference between HF and STD groups. § indicates significant statistical difference between HF and HF + MJ. The single symbol corresponds to p < .05; the double symbol to p < .01, and the triple symbol to p < .001 were  (Russo et al., 2011;Wang et al., 2017) did not report the presence of these compounds in the juice, but only in the C. reticulata flavedo (Wang et al., 2017;Zhang et al., 2014), while they were found in clementine cultivars (Sentandreu, Izquierdo, & Sendra, 2007).
The quantitative analysis (Table 4) revealed that compounds 9-11 and 13 were present in this juice only in traces, while 12 and 14 were more abundant, with 12 present in higher quantity than 14.
Among flavonoid glycosides compound 6, identified as hesperidin or its isomer neohesperidin, was found to be the most abundant compound in agreement with the literature (Russo et al., 2011;Wang et al., 2017), followed by compounds 11, 4, 7, and 8 (Table 4). Note: Compound numbers correspond to those listed in Table 3.