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: We thank the Postgraduate Program in Clinical and Experimental Pathophysiology for administrative and financial support.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers.

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Download references. The authors would like to thank Prof. Rashed Biochemistry Department, Faculty of Medicine, Cairo University, Egypt for assistance in performing PCR technique. Ahmed, Faculty of Veterinary Medicine, Zagazig University for histopathological examination of liver and A.

Tahrani, Institute of Pharmacy and Molecular Biotechnology, Heidelberg University, Germany for running LC-MS. Department of Pharmacology and Toxicology, Faculty of Pharmacy, Zagazig University, Zagazig, , Egypt. Department of Pharmacognosy, Faculty of Pharmacy, Zagazig University, Zagazig, , Egypt.

Institute of Pharmacy and Molecular Biotechnology, Heidelberg University, Im Neuenheimer Feld , , Heidelberg, Germany. Department of Pharmacology, Faculty of Pharmacy, University of Zagazig, Zagazig, , Egypt.

You can also search for this author in PubMed Google Scholar. Correspondence to Mona F. Reprints and permissions. Mahmoud, M. et al. Hepatoprotective effect of limonin, a natural limonoid from the seed of Citrus aurantium var.

bigaradia , on D-galactosamine-induced liver injury in rats. Naunyn-Schmiedeberg's Arch Pharmacol , — Download citation. Received : 01 July Accepted : 30 October