Effects of Pyridoxine on Selected Appetite Regulating Peptides mRNA Expression in Hypothalamic PVN / ARC Nuclei and Gastrointestinal Tract Tissues

An experiment was conducted to investigate the effect of dietary pyridoxine on the gene expression of appetite-regulating peptides in the hypothalamus and gastrointestinal tract of rabbits. Thirty-two rabbits were randomly divided into 2 treatments for 8 weeks (16 replicates/group and 1 rabbit/replicate). The treatments were fed a basal diet (control, measured pyridoxine content is 4.51 mg/kg) and the basal diet with a pyridoxine supplementation at 10 mg/kg (pyridoxine, measured pyridoxine content is 14.64 mg/kg). The results showed that dietary pyridoxine did not significantly alter the mRNA levels of neuropeptide Y, agouti related peptide, pro-opiomelanocortin and cocaine, amphetamine regulated transcript, peptide YY and cholecystokinin in arcuate nucleus, peptide YY in jejunum and ileum, and cholecystokinin in duodenum, jejunum and ileum (P > 0.05). Compared with the control, the mRNA levels of corticotropin-releasing hormone and melanocortin 4 receptor in paraventricular nuclei and peptide YY in duodenum were significantly decreased after pyridoxine treatment (P < 0.05). In conclusion, the appetite genes of melanocortin 4 receptor and corticotropin-releasing hormone in paraventricular nuclei and peptide YY in duodenum are involved in the pyridoxine-caused hyperphagia.


Introduction
The hypothalamus is a crucial region for integrating signal from central and peripheral pathways and plays a major role in appetite regulation.In the arcuate nucleus (ARC) of hypothalamus, there are 2 major neuronal populations, which influence energy homeostasis.One neuronal circuit inhibits food intake, via the expression of the pro-opiomelanocortin (POMC) and cocaine and amphetamine regulated transcript (CART) [1] [2].The other neuronal circuit stimulates food intake, via the expression of neuropeptide Y (NPY) and agouti related peptide (AgRP)  [3].The NPY/AgRP and POMC/CART neurones perform a primary sensory function in the neural regulation of appetite.These "first order" neurones then relay metabolic information to other "second order" neuronal populations located elsewhere in the hypothalamus such as the corticotropin-releasing hormone (CRH) and melanocortin 4 receptor (MC4R) in the paraventricular nuclei (PVN) [4].
Gut hormones act to modulate digestion and absorption of nutrients.However, they also act as neurotransmitters within the central nervous system to control food intake.Peptide YY3-36 (PYY) is secreted predominantly from the distal gastrointestinal tract, which suppress appetite through sending signals to the hypothalamus [5].Another brain-gut peptide is cholecystokinin (CCK), although which can not pass the blood brain barrier, can reduce feed intake though afferent vagal fibres to the caudal brainstem [6].
Pyridoxine (vitamin B 6 ) is essential for absorption and metabolism of amino acids and the development of red blood cells [7].Dietary pyridoxine can also alter the appetite.Low level of pyridoxine depressed appetite in chickens [8].Baby pigs with pyridoxine deficiency have poor appetite and growth [9].Dietary pyridoxine significantly attenuated the anorexia caused by heat stress in fingerlings [10].Our previous study also showed that dietary pyridoxine increased significantly food intake in a dose-dependent manner (0 to 20 mg/kg) in Rex rabbits [11].But the effect of pyridoxine appetite-related peptides is still unknown.Pyridoxine is important for development and function of the nervous system.It acts as coenzymes for transaminases and controls the biosynthesis of neurotransmitters (e.g., gamma-aminobutyric acid, dopamine, and serotonin) [12].Thus, we infer that the central and peripheric peptides may be regulated by the pyridoxine.In the present study, we investigated the effect of pyridoxine on the gene expression of food intake regulatory peptides in the hypothalamus and gastrointestinal tract of Rex rabbits.And the results will be useful to understand the progress of pyridoxine regulating energy homeostasis.

Animals
Three-month-old Rex rabbits were individually housed in self-made cages (60 cm × 40 cm × 40 cm).Temperature and lighting were maintained according to commercial conditions.The diets were formulated according to the values to growing from de Blas and Mateos [13] and pelleted by pressure.The diameter of the pellets was 4 mm.All rabbits received a diet containing 16.5% crude protein, 17% crude fiber and 10.5 MJ/kg of digestible energy.

Experimental Protocol and Sample Collection
At 90 days of age, 32 rabbits of similar body weight (1682 ± 40 g) were divided into 2 groups, with 16 replicates per group and 1 rabbit per replicate.Rabbits were randomly subjected to 1 of the following 2 treatments: feeding basal diet (control; measured pyridoxine content was 4.51 mg/kg) or feeding basal diet with additional 10 mg/kg pyridoxine supplementation (pyridoxine; measured pyridoxine content was 14.64 mg/kg).The dose of pyridoxine was according to the study by Liu et al. [11], which found the pyridoxine addition of 10 mg/kg in basal diet could significantly increase significantly food intake in Rex rabbits.
The experiment lasted for 8 weeks which included 1-week adaptation period and 7-week experimental period.At the end of the trial, rabbits were electrically stunned and killed [14].The ARC and PVN samples were collected according to the method described in Prior et al. [15] and Mano-Otagiri et al. [16].The samples of the whole intestinal tract were removed, and segments were taken from the midpoint of duodenum (5 cm from the pylorus), jejunum (50% along the small intestine) and ileum (5 cm from the ileocecal junction) [17].After being snap-frozen in liquid nitrogen, the tissue samples were stored at −80˚C until RNA extraction.

RNA Isolation and Analysis
Total RNA extraction and qRT-PCR were performed as described previously [18] [19].Sequences of primers are shown in Table 1.The PCR data were analyzed with the 2 −ΔΔCT method.The mRNA levels of target genes were normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA (ΔCT).On the basis of the Ct values, GAPDH mRNA levels were stable across the 2 treatments in this study (P > 0.1).

Statistical Analysis
The data are presented as the means ± SEM.Homogeneity of variances among the treatments was confirmed using Bartlett's test.All data were subjected to one-way ANOVA to test the main effect of the treatment.When the main effect of the treatment was significant, the differences between means were assessed by Duncan's multiple range analysis.P < 0.05 was considered statistically significant.

Results
Although no significant difference was observed in AgRP, NPY, POMC and CART mRNA levels (Figure 1(a)) in ARC between 2 groups (P > 0.05), CRH and MC4R mRNA levels in PVN were significantly decreased after pyridoxine  treatment (P < 0.05, Figure 1(b)).By detecting the PYY gene expression in different tissues of control rabbits, we found that the major expressed location is duodenum, and ARC had a lower expression of PYY (Figure 2).As shown in Figure 3(a), dietary addition of pyridoxine significantly decreased the PYY gene expression in duodenum (P < 0.05), but did not alter the PYY gene expression in ARC, jejunum and ileum (P > 0.05).Besides, dietary pyridoxine treatment did not significantly alter on CCK gene expression in all texted tissues (P > 0.05,

Discussion
Our previous study showed that pyridoxine could increase the food intake in rabbits [11], but the information in regards to that appetitive peptides response to this remains scarce.In the present experiment, the major appetite regulatory peptides in the hypothalamus (e.g., NPY, AgRP, POMC, CART, MC4R, CRH, CCK and PYY) and gastrointestinal tract (CCK and PYY) were detected after pyridoxine treatment in rabbits.
The balance between food intake and energy expenditure is regulated by the central nervous system, especially the hypothalamus.The dietary pyridoxine did not significantly affect the transcription of NPY/AgRP and POMC/CART neurons in the ARC in the present study.But Sanchez-Hernandez et al. [20] found   The MC4R are found in hypothalamic nuclei implicated in energy homeostasis, such as the ventromedial nucleus (VMH) and PVN [21].Lacking the type-4 melanocortin receptor leads to hyperphagia and obesity in rodents [22].And intracerebroventricular administration of a very low dose of melanocortin receptor agonist in adult rats reduces appetite [23].In our study, dietary pyridoxine treatment significantly decreased the MC4R gene expression in PVN, which suggests the MC4R may be an important target in pyridoxine-caused hyperphagia.The finding is congruent with the results of other vitamin study in human, which have low intake of folate and vitamin D, and have lower MC4R expression [24].
L. intake is inhibited after central CRH injection [25].The previous study found vitamin C supplementation decreased the synthesis of CRH in adrenalectomized rats and normal rats.Our results showed that dietary pyridoxine down-regulates the CRH mRNA levels in PVN, which implies that the CRH neurones participate in the process that pyridoxine affects appetite.Besides, the suppressed CRH mRNA levels by pyridoxine treatment may be associated with the MC4R.The MC4R mRNA is high expressed in CRH neurones, and activation of MC4R expression can increase CRH transcription [26].
Appetitive regulation is also involving peripheral control sites.The CCK is a gut peptide that has long been established to act as a postprandial satiety signal [27].Although CCK mRNA levels were increased in the duodenum and jejunum in response to fatty acids, glucose and amino acids [28] [29], the CCK gene expression is not sensitive to the dietary pyridoxine in our study.
Peptide YY3-36 can increase food intake if administered directly into the cerebrospinal fluid.In contrast, peripherally administered PYY3-36 can reduce food intake [28].The distribution of PYY is species diversity.In rabbits, the duodenum has a higher expression than other small intestines.But PYY is mainly expressed in ileum in mammals [30].The PYY can be stimulated by intraluminal nutrients, including glucose, bile salts, lipids, short-chain fatty acids and amino acids [31].Our results showed that dietary pyridoxine decreased PYY gene expression in duodenum, which is inconsistent with lipid-soluble vitamins-treated experiment that vitamin D increased circulating protein levels of PYY [32].Coll et al. [28] has proved that the anorexia effect of PYY was initially thought to be mediated through the central melanocortin system.Thus, the suppressive MC4R mRNA level of PVN in pyridoxine treatment may be related to decrease PYY levels in peripheral tissue.

Conclusion
The present study showed that dietary pyridoxine decreased the mRNA levels of MC4R and CRH in PVN and PYY in duodenum, but not alter the gene expression of AgRP, NPY, POMC and CART in ARC, PYY in ARC, jejunum and ileum and CCK in ARC, duodenum, jejunum and ileum.The results suggest that appetite-related genes of MC4R and CRH in PVN and PYY in duodenum may be involved in the pyridoxine-caused hyperphagia.

Figure 2 .
Figure 2. Peptide YY3-36 (PYY) gene expression in different tissue of control rabbits.Values are shown as the mean ± SE (n = 8).a, b Means with different letters above bars are significantly different (P < 0.05).

Table 1 .
Gene-specific primers used for the analysis of rabbit gene expression.
Liu et al.