Such modulation has a direct and beneficial effect on glucose and lipid metabolism, regardless of the effect on body weight 20 , In addition, natural compounds generally have fewer adverse effects aurantium comes from small fruit trees approximately five meters tall and with scented white flowers 22 belonging to the Rutaceae family and popularly known for bitter orange, sour orange, and Seville orange Due to their medicinal properties, products derived from C.

aurantium are commonly used as medicine and in dietary supplements In , the Food and Drug Administration FDA banned the use of ephedra, derived from Ephedra sinica , in dietary supplements in the United States due to its clinical association with heart and central nervous system problems Synephrine is a chiral amine that is present in nature in the form R — - -p-synephrine or l-synephrine 28 , 29 and a compound chemically similar to ephedrine, presenting with a similar structural composition and differing only by a hydroxyl ring in the para position of the benzene ring 30 and a methyl group on the side chain CH3 present in ephedrine As ephedrine, synephrine is a β3 adrenergic agonist receptor with thermogenic and lipolytic actions This structural variation alters the pharmacokinetics, resulting in fewer adverse effects on heart rate and blood pressure than ephedrine.

Thus, the use of synephrine as a substitute for ephedrine has become more frequent p-Synephrine is present in greater quantities in bitter orange fruit peel and is the main active component of C.

aurantium Citrus aurantium and synephrine are known for their therapeutic potential in thermogenesis stimulation. However, studies that indicate C. aurantium and synephrine as inducers of weight loss and thermogenic action in adipose tissue are still scarce Moreover, the most of them are carried out in combination with other medicines and plants or to assess its toxicity 28 , 34 , Considering all the aforementioned factors, we hypothesized that adolescence can be considered an important window of opportunity for the implementation of anti-obesity therapeutic strategies 18 , Therefore, new treatment alternatives that are more efficient for obesity management should be studied.

Here, we investigated the effectiveness of C. The sample size was calculated based on previous experimental findings that have robustly demonstrated statistically significant increases in biometric parameters, such as body mass and adiposity, relative to the control group In adherence to the principles of the 3 Rs model reduction, refinement, replacement , we also sought to minimize the utilization of animals while still preserving statistical significance.

Three-month-old male and female nulliparous mice were mated in a ratio for 7 days. After birth, the litters were adjusted to 9 pups per mother. To induce early overfeeding small litter group — SL , on postnatal Day 3 PND3 , the litter size was reduced to 3 pups per mother 18 mothers.

The control group normal litter group—NL was maintained with 9 pups per mother 12 mothers until weaning PND21 , when reduced to 6 animals per group. On PND21, the SL and NL groups were subdivided into 4 groups 10—12 animals per group :.

Only male mice were used in the whole experiment. All groups were treated with their respective doses administered by gavage, during PND30 to PND49, that correspond to the period of adolescence in mice 38 Supplementary Table 1.

All groups received the same volume through gavage ul. The doses of C. aurantium and synephrine were based on the descriptions by Deshmukh et al. The extracts of C. Isolated synephrine was obtained from Sigma Aldrich lot BCBW and code All 3 treated groups received the same amount of synephrine 1.

Figure 1. Experimental model. Postnatal day PND. Mice raised in normal litter NL ; Mice raised in normal litters treated with C. During the lactation period 21 days of life , the animals were weighed daily. After weaning, the animals were weighed every 3 days on a mini digital weight scale Professional digital weight scale MOD Body composition was analyzed using whole-body nuclear magnetic resonance NMR imaging Minispec LF90 TD-NMR, Bruker, Rheinstetten, Germany in the pretreatment and posttreatment periods to evaluate total fat mass.

The non-anesthetized animals were placed in a transparent plastic cylinder and kept immobile due to the insertion of a very tight plunger in the cylinder. Soon after, the cylinder with the animal was inserted into the NMR chamber, remaining during the examination for approximately 2 min.

The data were expressed in grams g of adipose mass. Systolic blood pressure, diastolic blood pressure, and heart rate were assessed using a non-invasive method Tail-cuff plethysmograph- LE Panlab, Barcelona, Spain.

The animals were acclimatized for 2 days, and then, the animals were submitted to the procedure again. The measurements were recorded and averaged. After 12 h of fasting, blood samples were collected to assess baseline glycaemia time 0. Glucose was measured at 15, 30, 60, and min after glucose administration.

At 50 days of age, after a 6-h fasting period — h , the animals were anesthetized with avertin 2,2,2—Tribromoethanol, 2-methylbutanol — 0.

The BAT was dissected, weighed, and prepared for morphological and molecular analysis real-time PCR. The adrenal glands were frozen to assess the adrenal catecholamine content.

The right adrenal tissue stored in acetic acid was used for analysis. The subsequent steps were performed as previously described From each tissue, non-serial sections 5 μm thick were obtained microtome Microtec-CUT , SC, USA.

Digital images were acquired randomly TIFF format using an Olympus DP71 camera coupled to an Olympus BX40 light microscope Olympus, Japan. Ten photomicrographs per animal were used. BAT digital images were analyzed, and their areas were calculated.

All photomicrographs were measured with Image-Pro Plus 5. Total RNA was extracted from BAT samples using the RNeasy Lipid Tissue kit Qiagen, Germantown, Maryland following the protocol described by the manufacturer. cDNA was synthesized using a reverse transcription kit Applied Biosystems Thermo Fisher Scientific, Massachusetts, USA , and the samples were incubated in a thermocycler Applied Biosystems Veriti 96 Well Thermal Cycler.

The primers were purchased from TaqMan Thermo Fisher Scientific Supplementary Table 2. In each reaction plate, the negative control without sample C- , the negative control without enzyme RT- , and the standard curve of serial dilution corresponding to the gene of interest were added.

The results were expressed in relation to the expression values of their control groups, which were 1 and normalized to the standard curve. Subsequently, we used these values for statistical analysis.

The efficiencies of each test were calculated from a serial dilution curve present on each plate, using only plates whose efficiencies were between 85 and Brown adipose tissue respiration was determined as previously described 40 , 41 with minor modifications.

BAT was prepared for measurements of respiratory flux rates by mechanic dissection with sharp forceps in relaxing buffer BIOPS; in mM: CaK2EGTA 2. After that, the interscapular brown adipose tissues were washed in ice-cold respiration medium MIR05—in mM: EGTA 0.

The respiratory rates of BAT were determined with the Oroboros 2k-Oxygraph Oroboros Instruments, Innsbruck, Austria in 2 ml of MIR05 at 37°C with continuous stirring.

Before adding the tissue into the chamber, wet weight measurements were taken, and a sample of 5—7 mg was used per chamber. All measurements were taken at oxygen concentrations above nmol ml-1 in the chamber. DatLab software Oroboros Instruments, Innsbruck, Austria was used for data acquisition and analysis.

Digitonin is used to permeabilize the cell membranes while leaving the mitochondrial membranes intact because of its specificity for solubilizing cholesterol, which exists in much higher concentrations in the plasma membrane.

The study was carried out with two groups of independent substrates in each chamber: chamber A, in mM glutamate 10, pyruvate 5, malate 2, ADP 1 and succinate 10, for the analysis of carbohydrate-related oxidation with electron entry through complexes I and II of the respiratory chain and chamber B, in mM palmitoyl-carnitine 0.

The addition of cytochrome c 10 μM allowed for the evaluation of the integrity of the mitochondrial membrane because an increase in respiration with the addition of cytochrome c indicates a defect in the outer mitochondrial membrane Statistical analyses were performed using GraphPad Prism software version 6.

The body weights of normal litters NL and small litters SL during lactation PND21 are shown in Figure 2. The small litter group SL had a higher body weight than the normal litter group NL on PND4 until weaning PND21, SL: 3.

NL: 2. Figure 2. Evolution of body weight during the lactation period 21 days of mice raised in normal NL and small SL litters. The SL group had a higher body weight NL: SL: SL: 3. aurantium and synephrine did not show significant differences in fat mass with the NL groups Figure 3D.

Figure 3. Effect of treatments with C. aurantium and synephrine on body weight A,C , body composition by Nuclear Magnetic Resonance B,D and tissue weight of visceral WAT E and BAT F of mice raised in normal and small litters in the pre-treatment A,B and post-treatment C—F period.

NL A,B. NL-Syn C,D. SL-Syn E,F. The accumulated body weight from PND30 to PND49 is depicted in Figure 4. The SL groups treated with C. Figure 4. Accumulated weight gain of mice raised in normal and small litters submitted to treatment with C. There was no significant difference in heart rate Supplementary Figure 1A , systolic blood pressure Supplementary Figure 1B , and diastolic blood pressure Supplementary Figure 1C in the normal and small litter groups treated with C.

aurantium and synephrine or vehicle. There was no significant difference in the OGTT Supplementary Figure 2A or the area under the curve AUC of the OGTT Supplementary Figure 2B in the normal and small litter groups treated with C.

The plasma concentration of leptin in the SL groups treated with C. aurantium and synephrine was not different from that in the NL groups or SL vehicle group Figure 5A. Figure 5. Effect of treatment with C. aurantium and synephrine in hormonal dosages. Plasma leptin A , Total T3 B and Free T4 C. There was no significant difference in the absolute catecholamine content in the adrenal gland Figure 6A.

There was no significant difference in plasma corticosterone Figure 6C. Figure 6. aurantium and synephrine on the medulla adrenal and plasma corticosterone. aurantium and synephrine 2-fold-increase vs. NL -Syn; 2-fold-increase vs.

Treatment with C. aurantium and synephrine was able to restore the lipid droplet size and quantity of nuclei in the small litter groups.

Figure 7. BAT histology by Hematoxylin—Eosin HE staining with 40× magnification. Quantitative analysis of lipid droplets and nucleus number of the BAT.

Figure 8 shows biomarkers related to thermogenesis in BAT. The NL groups did not show differences in gene expression in BAT. aurantium and synephrine showed increased gene expression of UCP-1, PRDM16, PGC-1α, and PPARγ.

Treatment of the SL group with C. SL groups treated with C. The SL-Syn group showed higher relative mRNA expression of PRDM than the SL and NL groups 2.

No significant difference was observed in the gene expression of CPT Figure 8D , ADRβ-3 Figure 8E , or BMP7 Figure 8G. However, treatment with C. Figure 8. aurantium and synephrine on thermogenic factors in BAT.

Also, SL-Syn group presented no changes in all parameters of BAT mitochondrial function evaluated. Figure 9. High resolution respirometry of brown adipose tissue from overfeed mice.

Flux per mass with substrates pyruvate, glutamate, malate, and succinate A. Flux per mass with substrates palmitoyl-L-carnitine, malate, and ADP B. It is well known that overfeeding early in life causes metabolic effects in the short- and long-term, but such effects are poorly investigated in adolescence.

The reduction in litter size is an effective and reproducible model of obesity 12 , 15 , Our results demonstrated that both overweight and metabolic changes typical of obesity persist from lactation to adolescence. Moreover, the administration of Citrus aurantium or its active compound, synephrine, proved efficacious in ameliorating certain metabolic dysfunctions induced by postnatal overfeeding, employing distinct mechanisms.

Conceicao et al. We find at PND30, overweight and high body fat in SL group by body composition NMR analyses. Furthermore, we showed that the SL group showed higher body weight from PND4 until adolescence.

Studies were carried out upon utilization of products derived from medicinal plants and nutraceuticals for the management of obesity and metabolic disorders Until this moment, no studies have demonstrated the effects of C.

aurantium and synephrine at the same doses and period adolescence used in the current investigation. Here, we used a smaller dose than the one usually used in other studies because it would not be interesting to use elevate adrenergic agonist dose in adolescent animals. Hansen and collaborators 30 demonstrated, in adult female rats, that the administration of C.

In agreement, we also observed that the SL group treated with C. aurantium or synephrine did not show significant differences in body weight and body fat on PND Synephrine, due to it is an adrenergic agonist effect and its similarity to ephedrine, has potential adverse effects upon the cardiovascular system However, we did not show changes in heart rate or systolic or diastolic blood pressure in the treatments with C.

aurantium extracts and synephrine. Citrus aurantium has been associated with improvement in hyperglycemia Although obese, the SL group had no changes in glucose homeostasis, with no significantly different in TOTG compared to the NL group.

Litter size reduction programming impairs leptin signaling and causes leptin resistance at PND 37 , Corroborating these findings, the SL group showed hyperleptinemia, indicating leptin resistance in these animals as early as 50 days old. However, C. aurantium and synephrine slightly reduced this hyperleptinemia induced by overfeeding.

In the literature, no studies were found about the effect of C. aurantium and synephrine on leptin secretion and signaling. Thus, precocious treatment with C. aurantium may prevent those animals from developing future glucose intolerance since leptin resistance can be one contributor factor to insulin resistance.

Rodrigues and collaborators 37 showed that, in PND21, the SL group had high TSH and serum thyroid hormone concentrations. However, on PND, this group showed normal TSH and lower T3 and T4 in serum. Here, treatment with C. aurantium increased total T3 and free T4 compared to all NL groups in this study, and synephrine SL-Syn increased even more because it was higher than in the SL group.

As there are no studies focusing the interplay between thyroid function and C. aurantium or synephrine , more studies are needed to better understand the mechanisms involved. aurantium , other than synephrine, can increase adrenal catecholamines, which can occur either by greater synthesis or by accumulation due to deficient secretion.

We measured only tissue catecholamines, and this increase was not enough to alter cardiovascular parameters. However, this change may have resulted in a slight increase in circulating adrenaline, inducing lipolysis in white and brown adipocytes, through their interaction with β-3 adrenergic receptors Only one in vitro study has reported the influence of C.

aurantium on the differentiation and activation of brown adipocytes through anti-adipogenic and thermogenic mechanisms In the current study, we explored the in vivo anti-obesity potential of C. aurantium extracts and its main active component, synephrine, in brown adipose tissue dysfunction of adolescent mice programmed by early postnatal overfeeding.

The whitening of BAT occurs in obesity. In this process, BAT has increased tissue mass with large lipid droplets, low vascularization, high pro-inflammatory cytokine expression, and low UCP-1 and other thermogenesis marker expression 13 , 48 , causing dysfunction.

In the qualitative and quantitative analyses of BAT, we demonstrated a reduction in its mass and in the content of lipid vesicles and an increase in the number of nuclei in the SL groups treated with C.

aurantium and synephrine, showing that the treatments normalized the BAT structure and recovered its original phenotype. We investigated some important markers of thermogenesis and activity in BAT, such as UCP-1, PRDM16, PCG-1α, CPT-1, beta-3AR, PPARγ, and BMP-7 Furthermore, we found, in general, higher gene expression of UCP1, PRDM 16, PGC-1α, and PPARγ, demonstrating the thermogenic action of both C.

aurantium and its active compound, synephrine. β-3 adrenergic receptor expression in BAT was unchanged, but we cannot ignore the effect of synephrine β-3 adrenergic ligand on adrenergic receptors.

PPARγ is responsible for positively regulating genes involved in lipid oxidation CPT-1 and thermogenesis, such as UCP-1 and PGC-1α responsible for mitochondrial biogenesis and stimulation of UCP-1 gene expression In addition, PPARγ participates in several physiological functions, such as glucose metabolism control, adipocyte differentiation regulation, and inflammatory response regulation.

This receptor is considered a primary regulator of adipogenesis, controlling cell differentiation of preadipocytes into mature adipocytes 51 and acting in the direction of white and brown adipocyte differentiation UCP-1 is a key marker of thermogenesis in BAT.

In experimental models, the absence of UCP-1 is associated with increased body weight and decreased thermogenesis in BAT In our model of obesity induced by litter size reduction, we observed BAT dysfunction and UCP-1 gene expression reduction. aurantium on BAT was better than synephrine alone that only caused a higher gene expression for PRDM16, a known transcriptional co-regulator capable to directing brown adipogenesis.

Mitochondria primarily produce ATP for cellular energy by directing proton flow to ATP synthase. This occurs when protons, temporarily stored in the intermembrane space, are released into the mitochondrial matrix by uncoupling proteins, reducing the membrane potential, and producing heat rather than ATP Thus, the induced decrease in proton flux is mediated by an increase in UCP1 in adipose tissue, increasing heat production and potentiating weight loss.

This mechanism is important as a therapeutic target in the regulation of body weight. Considering the higher expression of UCP1 in BAT, this could be one of the mechanisms of action of C.

UCP-1 activity is mainly regulated by fatty acids that represent the main energy substrates for oxidation during thermogenesis, providing NADH and FADH2 to supply the respiratory chain and ensure the proton gradient Isolated synephrine, in turn, did not change oxygen consumption in relation to the energy substrates studied.

In general, C. aurantium and synephrine increased thermogenic marker expression in BAT without modifying the cardiovascular system.

The modern obesogenic environment can significantly increase obesity worldwide, especially in adolescence This period is a crucial stage of human development when several psychological and social changes occur in addition to the acquisition of new life habits that are the foundation for health and wellbeing in adulthood Our results indicate that obesity treatment in the critical period of adolescence, when the neurological system is particularly sensitive to interventions, can be a good therapeutic strategy.

Even when using C. aurantium or synephrine at a low dose, both showed beneficial effects, reducing adipose tissue mass, and improving BAT functionality without impacting blood pressure and glucose homeostasis. With due care in extrapolating therapy from animals to humans, the set of our findings suggest that therapeutic use of these compounds can improve the metabolic profile of obese adolescents without adverse cardiovascular side effects.

Further studies are necessary to better evaluate the safety of using both C. The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

The animal study was approved by the Animal Care and Use Committee of the Biology Institute of the State University of Rio de Janeiro. The study was conducted in accordance with the local legislation and institutional requirements.

AG: Conceptualization, Formal analysis, Investigation, Writing — original draft. This work was supported by the Carlos Chagas Filho Foundation for Research Support of the State of Rio de Janeiro FAPERJ , grant numbers: The potential athletic benefits are as follows:.

Non — Athletic Benefits of Citrus Aurantium:. Citrus aurantium may be beneficial in the following conditions:. Citrus Aurantium Triple Paradox:. Depending on whether you take extracts of the leaves or peels of the immature or mature fruits, citrus aurantium shows three paradox effects:.

Dosage and Interactions:. The recommended dosage is — mg of the extract. Similar to grapefruit, citrus aurantium interacts with the medications that are metabolized by the enzyme complex called cytochrome P Citrus aurantium should be avoided in the following conditions:.

Log in Register. Don't have an account yet? Register now! Remember Me. Citrus Aurantium: Also known as bitter orange , Seville orange , zhi chi , and chongcao , citrus aurantium is a member of citrus trees and its fruits and leaves have been used for medicinal and athletic purposes.

The peels of bitter orange contain synephrine, octopamine, tyramine, hordenine, N-methyltyramine, volatile oils , and carotenoids. Athletic Benefits of Citrus Aurantium: Since ephedrine has been banned in sports, synephrine — containing citrus aurantium may be a good alternative.

The potential athletic benefits are as follows: May improve athletic performance by acting as a mild stimulant. Promotes mental clarity. Helps promote athletic agility.

Useful in weight loss by decreasing appetite and increasing basal metabolic rate BMR.