Orexin A but not orexin B regulates lipid metabolism and leptin secretion in isolated porcine adipocytes

https://doi.org/10.1016/j.domaniend.2017.12.003Get rights and content

Highlights

  • Orexin's receptors are expressed in porcine adipose tissue as well as in isolated porcine adipocytes.

  • Orexin A controls the intensity of lipogenesis, lipolysis, and glucose uptake in isolated porcine adipocytes.

  • Orexin A stimulates leptin mRNA expression and secretion in porcine adipocytes.

  • Orexin A increases mRNA and protein expression of glucose transporter 4.

  • Orexin A controls cell viability of isolated porcine adipocytes via extracellular signal-regulated kinase 1 and 2.

Abstract

It is well known that orexins are involved in the metabolism and endocrine function of rodent adipocytes, but there are no data on other animal species, including pigs. Therefore, in this study, we tested the hypothesis that orexin A (OxA) and orexin B (OxB) modulate the metabolism and endocrine functions of isolated porcine adipocytes and adipose tissue explants. Moreover, we characterized the possible mechanism of OxA action in porcine adipocytes. According to the results, both orexin receptor 1 and orexin receptor 2 were expressed in the porcine adipose tissue. We found that OxA suppressed the release of glycerol from porcine adipocytes both in the absence (basal lipolysis; P < 0.05) and in the presence (stimulated lipolysis; P < 0.05) of isoproterenol. Orexin A increased basal and insulin-stimulated glucose uptake (P < 0.05), as well as it enhanced the rate of glucose incorporation into lipids with insulin (stimulated lipogenesis; P < 0.01) or without insulin (basal; P < 0.05). We have also shown that OxA stimulated the mRNA expression of glucose transporter 4 (P < 0.05) and its translocation into the plasma membrane (P < 0.01). Moreover, OxA upregulated the mRNA expression of leptin in isolated porcine adipocytes (P < 0.05) and increased the secretion of leptin (P < 0.05). We have also demonstrated one of the possible mechanisms of OxA action in adipocytes. In the presence of extracellular-signal-regulated kinase 1 and 2 (ERK1/2) inhibitor, the effect of OxA was not detectable in porcine adipocytes, which indicates that this peptide increased cell viability via ERK1/2 pathway (P < 0.05). However, OxB did not show any effect on the metabolism and endocrine functions of porcine adipocytes. In summary, we have shown for the first time that OxA has a significant impact on the intensity of lipolysis, glucose uptake, lipogenesis, as well as on the expression and secretion of leptin. Therefore, we conclude that OxA but not OxB regulates lipid metabolism in porcine adipose tissue and that this regulation is partly mediated via ERK1/2 pathway. The action of orexins should be further explored to better understand their role in the regulation of adiposity in pigs.

Introduction

Neuropeptides orexin A (OxA) and orexin B (OxB), also known as hypocretin 1 and hypocretin 2, respectively, were discovered in 1998 [1], [2]. These 2 isoforms are derived from a common precursor, preproorexin. Biological activity of orexins is regulated by 2 G protein-coupled receptors, namely, orexin receptor 1 (OxR1) and orexin receptor 2 (OxR2).

The knowledge about the role of orexins in regulation of metabolism was focused on human and laboratory animals. However, localization and functions of orexins system in domestic animals have been also investigated. It is known that in different species of livestock (pigs, cattle, horse, and sheep) both orexins and their receptors are expressed in the nervous system and different brain regions including hypothalamus, pituitary gland, preoptic area, stalk median eminence, hippocampus, amygdala, olfactory bulb, and pineal gland [3], [4], [5]. Moreover, the presence of orexins and their receptors has been detected in reproductive system, gastrointestinal tract, pancreas, kidney, and adrenal gland [5], [6].

This widespread distribution of orexins system indicates that OxA and OxB are involved in a number of biological functions in body of farm animals. Literature data show that these peptides regulate food intake [7], [8]. They also influence pancreas and regulate insulin [9] and glucagon secretion [10]. Furthermore, OxA and OxB have been shown to regulate the estrous cycle and gonadal tract by influencing GnRH and LH secretion, and ovarian, uterine, and testis steroidogenesis in pigs [5], [11], [12].

It is well established in rodents that orexins influenced on metabolic processes of adipose tissue [13], [14]. However, there are no data regarding OxA and OxB action in adipose tissue of pigs. Therefore, in this study, we tested the hypothesis that OxA and OxB modulate the metabolism and endocrine functions of isolated porcine adipocytes and adipose tissue explants. Pigs are a very important species of livestock worldwide, and it can be used as an animal model for the study of metabolism of adipose tissue in other species [15]. Specifically, we focused our attention on changes of intensity of lipolysis, glucose uptake, and lipogenesis as well as leptin expression and secretion in isolated porcine adipocytes under the influence of OxA and OxB. We further hypothesized that orexins regulate lipid metabolism in porcine adipose tissue partly via extracellular-signal-regulated kinase 1 and 2 (ERK1/2) pathway.

The results of this study can help to understand the effects of these peptides in domestic animals which may contribute to the development of new strategy of animal nutrition and production programs.

Section snippets

Animals

The study and all trial procedures were approved by the Local Ethical Committee No. 10 of Poznań, Poland (approval number 10/2011).

Six 21-day-old male Złotnicka piglets (average body weight of 6.83 ± 0.79 kg) from an Experimental Station of the Poznan University of Life Sciences, Złotniki, Poland, were sacrificed to obtain adipose tissue. Animals were treated by sedation (using medetomidine hydrochloride and ketamine) and then killed by exsanguination. Experiments were conducted within 30 min

Orexins and OxR expression in porcine-isolated adipocytes and adipose tissue

First, we decided to investigate the mRNA and protein expression level of orexin receptors in visceral and subcutaneous adipose tissue as well as in isolated porcine adipocytes (Fig. 1A–C). We found that there was a higher level of mRNA expression of OxR1 than that of OxR2 in isolated fat cells and examined adipose tissue depots (Fig. 1A; P < 0.01). We also found that OxR1 and OxR2 proteins were expressed in all tested tissues and isolated cells. Despite the use of the same amounts of protein,

Discussion

One of the biggest challenges in porcine nutrition is increasing the productivity and improving the meat quality by regulating fat content in pigs. Despite that the role of orexins in porcine fat metabolism has been elucidated, there is limited information on the regulatory influence of orexins on the metabolism of peripheral tissues in pigs. Hypothalamic expression of orexins and their receptor depends on weaning time and weaning pig weight [21]. Moreover, research has shown that in sheep, the

Acknowledgments

This study was supported by the Ministry of Sciences and Higher Education/National Science Centre, Poland, grant NN311508339.

E. P. O. and P. A. K., designed the study, obtained the data and wrote the manuscript. K. W. N., contributed to the study design, edited, supported and critically revised the manuscript, and contributed to the discussion. M. S., R. M., D. S. Z., and P. K. obtained the data, collected and analyzed the data, and revised the manuscript. All authors have given final approval

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