Integrative proteomic and phosphoproteomic analysis in the female goat hypothalamus to study the onset of puberty.

BACKGROUND: Puberty marks the end of childhood and achieve sexual maturation and fertility. The role of hypothalamic proteins in regulating puberty onset is unclear. We performed a comprehensive differential proteomics and phosphoproteomics analysis in prepubertal and pubertal goats to determine the roles of hypothalamic proteins and phosphoproteins during the onset of puberty. RESULTS: We used peptide and posttranslational modifications peptide quantification and statistical analyses, and identified 69 differentially expressed proteins from 5,057 proteins and 576 differentially expressed phosphopeptides from 1574 phosphorylated proteins. Combined proteomic and phosphoproteomics, 759 correlated proteins were identified, of which 5 were differentially expressed only at the protein level, and 201 were only differentially expressed at the phosphoprotein level. Pathway enrichment analyses revealed that the majority of correlated proteins were associated with glycolysis/gluconeogenesis, Fc gamma R-mediated phagocytosis, focal adhesion, GABAergic synapse, and Rap1 signaling pathway. These pathways are related to cell proliferation, neurocyte migration, and promoting the release of gonadotropin-releasing hormone in the hypothalamus. CTNNB1 occupied important locations in the protein-protein interaction network and is involved in focal adhesion. CONCLUSION: The results demonstrate that the proteins differentially expression only at the protein level or only differentially expressed at the phosphoprotein level and their related signalling pathways are crucial in regulating puberty in goats. These differentially expressed proteins and phosphorylated proteins may constitute the proteomic backgrounds between the two different stages.

Knockdown of lncRNA Meg3 delays the onset of puberty in female rats.

This study investigated how lncRNA Meg3 affects the onset of puberty in female rats. We determined Meg3 expression in the hypothalamus-pituitary-ovary axis of female rats at the infancy, prepubertal, pubertal, and adult life stages, using quantitative reverse transcription polymerase chain reaction (qRT-PCR). We also assessed the effects of Meg3 knockdown on the expression levels of puberty-related genes and Wnt/ß-catenin proteins in the hypothalamus, time of puberty onset, levels of reproductive genes and hormones, and ovarian morphology in female rats. Meg3 expression in the ovary varied significantly between prepuberty and puberty (P < 0.01). Meg3 knockdown decreased the expression of Gnrh, and Kiss1 mRNA (P < 0.05) and increased the expression of Wnt (P < 0.01) and ß-catenin proteins (P < 0.05) in the hypothalamic cells. Onset of puberty in Meg3 knockdown rats was delayed compared to the control group (P < 0.05). Meg3 knockdown decreased Gnrh mRNA levels (P < 0.05) and increased Rfrp-3 mRNA levels (P < 0.05) in the hypothalamus. The serum concentrations of progesterone (P4) and estradiol (E2) of Meg3 knockdown rats were lower than those in the control animals (P < 0.05). Higher longitudinal diameter and ovary weight were found in Meg3 knockdown rats (P < 0.05). These findings suggest that Meg3 regulates the expression of Gnrh, Kiss-1 mRNA and Wnt/ß-catenin proteins in the hypothalamic cells, and Gnrh, Rfrp-3 mRNA of the hypothalamus and the serum concentration of P4 and E2, and its knockdown delays the onset of puberty in female rats.

Analysis of protein phosphorylation sites in the hypothalamus tissues of pubescent goats.

Protein phosphorylation plays an important role in animal reproduction. However, its role in the onset of puberty in goats remains largely unexplored. Accordingly, in the present study, the molecular changes controlling the onset of puberty in goats were investigated by identifying the differentially phosphorylated proteins (DPPs) and phosphorylation sites (DPSs) in the hypothalami of prepubertal and pubertal female goats using LC-MS/MS and tandem mass tag labelling. A total of 3265 phosphopeptides corresponding to 1628 phosphoproteins were identified, including 234 upregulated and 342 downregulated phosphopeptides. The DPSs HTT, MAP1B, CAMKK1, MAP2, DNAJC5, and GAP43 were identified. These DPPs are enriched in the endocytosis, cAMP signaling, Rap1 signaling, melanogenesis, and insulin secretion pathways. These pathways are related to gonadotropin-releasing hormone and puberty. In particular, glucose-6-phosphate isomerase, fructose-bisphosphate aldolase C, and fructose-bisphosphate aldolase A occupy important locations in the protein-protein interaction network. These data provide evidence for a complex interaction network in goat hypothalamus proteins that affects puberty. Furthermore, they may help identify new puberty-regulating candidates and/or serve as an important resource for exploring the physiological mechanism of puberty onset in mammals. SIGNIFICANCE: This study provides evidence for a complex interaction network in goat hypothalamus proteins that affects puberty. Furthermore, our data may help identify new puberty-regulating candidates and/or serve as an important resource for exploring the physiological mechanism of puberty onset in mammals.

Proteomic analysis of hypothalamus in prepubertal and pubertal female goat.

The functions of proteins at the onset of puberty in goats remain largely unexplored. To identify the proteins regulating puberty in goats, we analysed protein abundance and pathways in the hypothalamus of female goats. We applied tandem mass tag (TMT) labelling, liquid chromatography-tandem mass spectrometry (LC-MS/MS), and parallel reaction monitoring (PRM) to examine hypothalamus of pubertal (cases; n = 3) and prepubertal (controls; n = 3) goats. We identified 5119 proteins, including 69 differentially abundant proteins (DAPs), of which 35 were upregulated and 34 were downregulated. Fourteen DAPs were randomly selected to verify these results using PRM, and the results were consistent with the TMT quantitative results. DAPs were enriched in MAPK signalling pathway, Ras signalling pathway, Autophagy-animal, Endocytosis, and PI3K/Akt/mTOR signalling pathway categories. These pathways are related to embryogenesis, cell proliferation, cell differentiation, and promoting the release of gonadotropin-releasing hormone (GnRH) in the hypothalamus. In particular, PDGFRß and MAP3K7 occupied important locations in the protein-protein interaction network. The results demonstrate that DAPs and their related signalling pathways are crucial in regulating puberty in goats. However, further research is needed to explore the functions of DAPs and their pathways to provide new insights into the mechanism of puberty onset. SIGNIFICANCE: In domestic animals, reaching the age of puberty is an event that contributes significantly to lifetime reproductive potential. And the hypothalamus functions directly in the complex systemic changes that control puberty. Our study was the first TMT proteomics analysis on hypothalamus tissues of pubertal goats, which revealed the changes of protein and pathways that are related to the onset of puberty. We identified 69 DAPs, which were enriched in the MAPK signaling pathway, the Ras signaling pathway, and the IGF-1/PI3K/Akt/mTOR pathway, suggesting that these processes were probably involved in the onset of puberty.

Knockdown of Ptprn-2 delays the onset of puberty in female rats.

In the present study, we evaluated how Ptprn-2 (encoding tyrosine phosphatase, receptor type, N2 polypeptide protein) affects the onset of puberty in female rats. We evaluated the expression of Ptprn-2 mRNA and protein in the hypothalamus-pituitary-ovary axis of female rats using real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) and immunofluorescence at infancy, prepuberty, puberty, peripuberty, and adulthood. We evaluated the effects of Ptprn-2 gene knockdown on different aspects of reproduction-related biology in female rats, including the expression levels of puberty-related genes in vivo and in vitro, the time to onset of puberty, the concentration of serum reproductive hormones, the morphology of ovaries, and the ultrastructure of pituitary gonadotropin cells. Our results demonstrated that PTPRN-2 was primarily distributed in the arcuate nucleus (ARC), periventricular nucleus (PeN), adenohypophysis, and the ovarian follicular theca, stroma, and granulosa cells of female rats at various stages. Ptprn-2 mRNA levels significantly varied between peripuberty and puberty (P < 0.05) in the hypothalamus and pituitary gland. In hypothalamic cells, Ptprn-2 knockdown decreased the expression of Ptprn-2 and Rfrp-3 mRNA (P < 0.05) and increased the levels of Gnrh and Kiss-1 mRNA (P < 0.05). Ptprn-2 knockdown in the hypothalamus resulted in delayed vaginal opening compared to the control group (n = 12, P < 0.01), and Ptprn-2, Gnrh, and Kiss-1 mRNA levels (P < 0.05) all decreased, while the expression of Igf-1 (P < 0.05) and Rfrp-3 mRNA (P < 0.01) increased. The concentrations of FSH and P4 in the serum of Ptprn-2 knockdown rats were lower than in control animals (P < 0.05). Large transverse perimeters and longitudinal perimeters (P < 0.05) were found in the ovaries of Ptprn-2 knockdown rats. There were fewer large secretory particles from gonadotropin cells in adenohypophysis tissue of the Ptprn-2 knockdown group compared to the control group. This indicates that Ptprn-2 knockdown can regulate levels of Gnrh, Kiss-1, and Rfrp-3 mRNA in the hypothalamus, regulate the concentration of serum FSH and P4, and alter the morphology of ovarian and gonadotropin cells, delaying the onset of puberty in female rats.

Black and Green Tea Supplements Ameliorate Male Infertility in a Murine Model of Obesity.

Obesity, a chronic metabolic disorder, can affect male reproductive function. As a functional beverage, tea has many biological activities and potential in the treatment of obesity. However, its effects on male reproductive damage induced by obesity are not yet clear. In this study, a murine model of obesity was established by feeding with high-fat diet (HF). A total of 24 male mice were divided into four groups: normal diet (control), HF, HF supplemented with 5% green tea powder (HF+G), and HF supplemented with 5% black tea powder (HF+B). The results showed that the HF + B significantly reduced the mouse body weight gain and testicular coefficient and lowered the serum insulin and leptin levels compared with the HF group. The sperm malformation rate of mice in the HF group had a significant increase when compared with the control group, the HF + B group had a significant decrease compared with the HF group, and no difference from the control group. The HF + G and HF + B significantly increased testosterone levels in serum compared with the HF group. The testosterone production-related gene cytochrome P450 family 11 subfamily a member (CYP11A1) and cytochrome p450 family 17 subfamily a member 1 (CYP17A1) expressions in testis were significantly increased in the HF + G group compared with HF group. In addition, the HF + G and HF + B abolished the effects of HF on superoxide dismutase (SOD), malondialdehyde, and glutathione levels in testis and antioxidant-related gene expressions of XRCC1 and SOD1. Overall, our findings have provided evidence that black and green tea has a positive effect on reducing reproductive damage in a male murine model of obesity, and that black tea is more effective than green tea.

Semantic Fisher Scores for Task Transfer: Using Objects to Classify Scenes.

The transfer of a neural network (CNN) trained to recognize objects to the task of scene classification is considered. A Bag-of-Semantics (BoS) representation is first induced, by feeding scene image patches to the object CNN, and representing the scene image by the ensuing bag of posterior class probability vectors (semantic posteriors). The encoding of the BoS with a Fisher vector (FV) is then studied. A link is established between the FV of any probabilistic model and the Q-function of the expectation-maximization (EM) algorithm used to estimate its parameters by maximum likelihood. This enables 1) immediate derivation of FVs for any model for which an EM algorithm exists, and 2) leveraging efficient implementations from the EM literature for the computation of FVs. It is then shown that standard FVs, such as those derived from Gaussian or even Dirichlet mixtures, are unsuccessful for the transfer of semantic posteriors, due to the highly non-linear nature of the probability simplex. The analysis of these FVs shows that significant benefits can ensue by 1) designing FVs in the natural parameter space of the multinomial distribution, and 2) adopting sophisticated probabilistic models of semantic feature covariance. The combination of these two insights leads to the encoding of the BoS in the natural parameter space of the multinomial, using a vector of Fisher scores derived from a mixture of factor analyzers (MFA). A network implementation of the MFA Fisher Score (MFA-FS), denoted as the MFAFSNet, is finally proposed to enable end-to-end training. Experiments with various object CNNs and datasets show that the approach has state-of-the-art transfer performance. Somewhat surprisingly, the scene classification results are superior to those of a CNN explicitly trained for scene classification, using a large scene dataset (Places). This suggests that holistic analysis is insufficient for scene classification. The modeling of local object semantics appears to be at least equally important. The two approaches are also shown to be strongly complementary, leading to very large scene classification gains when combined, and outperforming all previous scene classification approaches by a sizable margin.

Inhibition of Autophagy Attenuated Intestinal Injury After Intestinal I/R via mTOR Signaling.

BACKGROUND: Intestinal ischemia/reperfusion (I/R) is a grave condition related to high morbidity and mortality. Autophagy, which can induce a new cell death named type II programmed cell death, has been reported in some intestinal diseases, but little is known in I/R-induced intestinal injury. In this study, we aimed to explore the role of autophagy in intestinal injury induced by I/R and its potential mechanisms. MATERIALS AND METHODS: The rats pretreated with rapamycin or 3-methyladenine had intestinal I/R injury. After reperfusion, intestinal injury was measured by Chiu's score, intestinal mucosal wet-to-dry ratio, and lactic acid level. Intestinal mucosal oxidative stress level was measured by malondialdehyde and superoxide dismutase. Autophagosome, LC3, and p62 were detected to evaluate autophagy level. Mammalian target of rapamycin (mTOR) was detected to explore potential mechanism. RESULTS: Chiu's score, intestinal mucosal wet-to-dry ratio, lactic acid level, malondialdehyde level, autophagosomes, and LC3-II/LC3-I were significantly increased, and superoxide dismutase level and expression of p62 were significantly decreased in intestinal mucosa after intestinal ischemia/reperfusion. Pretreatment with rapamycin significantly aggravated intestinal injury evidenced by increased Chiu's score, intestinal mucosal wet-to-dry ratio and lactic acid level, increased autophagy level evidenced by increased autophagosomes and LC3-II/LC3-I and decreased expression of p62, and downregulated expression of p-mTOR/mTOR. On the contrary, pretreatment with 3-methyladenine significantly attenuated intestinal injury and autophagy level and upregulated expression of p-mTOR/mTOR. CONCLUSIONS: In summary, autophagy was significantly enhanced in intestinal mucosa after intestinal ischemia/reperfusion, and inhibition of autophagy attenuated intestinal injury induced by I/R through activating mTOR signaling.

Identification of differential genomic DNA Methylation in the hypothalamus of pubertal rat using reduced representation Bisulfite sequencing.

BACKGROUND: There are many variables affecting the onset of puberty in animals, including genetic, nutritional, and environmental factors. Recent studies suggest that epigenetic regulation, especially DNA methylation, plays a majorrole in the regulation of puberty. However, there have been no reports on DNA methylation of the pubertal genome. METHODS: We investigated DNA methylation in the female rat hypothalamus at prepuberty and puberty using reduced representation bisulfite sequencing technology. The identified genes and signaling pathways exhibiting changes to DNA methylation in pubertal rats were determined by Gene Ontogeny and Kyoto Encyclopedia of Genes and Genomes analysis. RESULTS: The distribution of the three types of methylated C bases in promoter and CpG island (CGI) regions in the hypothalamus was as follows: 87.79% CG, 3.05% CHG, 9.16% CHH for promoters, and 88.35% CG, 3.21% CHG, 88.35% CHH for CGI in prepubertal rats; and 90.78% CG, 2.13% CHG, 7.09% CHH for promoters, and 88.59% CG, 88.59% CHG, 8.35% CHH for CGI in pubertal animals. CG showed the highest percentage of methylation, and was the highest methylation state in CGI. Compared to prepubertal hyoyhalamus samples, we identified ten genes with altered methylation in promoter regions in the pubertal hypothalamus samples, and 43 genes with altered methylation in the CGI. Changes in DNA methylation were found in gonadotropin-releasing hormone signaling pathways, and the oocyte meiosis pathway. CONCLUSION: Our results demonstrate changes in DNA methylation occur in female rats from prepuberty to puberty suggestng DNA methylation may play a crucial role in the regulation of puberty onset. This study provides essential information for future studies on the role of epigenetics in the regulation of puberty.

DNA Methylation Patterns in the Hypothalamus of Female Pubertal Goats.

Female pubertal development is tightly controlled by complex mechanisms, including neuroendocrine and epigenetic regulatory pathways. Specific gene expression patterns can be influenced by DNA methylation changes in the hypothalamus, which can in turn regulate timing of puberty onset. In order to understand the relationship between DNA methylation changes and gene expression patterns in the hypothalamus of pubertal goats, whole-genome bisulfite sequencing and RNA-sequencing analyses were carried out. There was a decline in DNA methylation levels in the hypothalamus during puberty and 268 differentially methylated regions (DMR) in the genome, with differential patterns in different gene regions. There were 1049 genes identified with distinct expression patterns. High levels of DNA methylation were detected in promoters, introns and 3'-untranslated regions (UTRs). Levels of methylation decreased gradually from promoters to 5'-UTRs and increased from 5'-UTRs to introns. Methylation density analysis demonstrated that methylation level variation was consistent with the density in the promoter, exon, intron, 5'-UTRs and 3'-UTRs. Analyses of CpG island (CGI) sites showed that the enriched gene contents were gene bodies, intergenic regions and introns, and these CGI sites were hypermethylated. Our study demonstrated that DNA methylation changes may influence gene expression profiles in the hypothalamus of goats during the onset of puberty, which may provide new insights into the mechanisms involved in pubertal onset.

Role of Nesfatin-1 in the Reproductive Axis of Male Rat.

Nesfatin-1 is an important molecule in the regulation of reproduction. However, its role in the reproductive axis in male animals remains to be understood. Here, we found that nesfatin-1 was mainly distributed in the arcuate nucleus (ARC), paraventricular nucleus (PVN), periventricular nucleus (PeN), and lateral hypothalamic area (LHA) of the hypothalamus; adenohypophysis and Leydig cells in male rats. Moreover, the concentrations of serum nesfatin-1 and its mRNA in hypothalamo-pituitary-gonadal axis (HPGA) vary with the age of the male rat. After intracerebroventricular injection of nesfatin-1, the hypothalamic genes for gonadotrophin releasing hormone (GnRH), kisspeptin (Kiss-1), pituitary genes for follicle-stimulate hormone ß(FSHß), luteinizing hormone ß(LHß), and genes for testicular steroidogenic acute regulatory (StAR) expression levels were decreased significantly. Nesfatin-1 significantly increased the expression of genes for 3ß-hydroxysteroid dehydrogenase (3ß-HSD), 17ß-hydroxysteroid dehydrogenase (17ß-HSD), and cytochrome P450 cleavage (P450scc) in the testis of pubertal rats, but their levels decreased in adult rats (P < 0.05), along with the serum FSH, LH, and testosterone (T) concentrations. After nesfatin-1 addition in vitro, T concentrations of the supernatant were significantly higher than that in the control group. These results were suggestive of the role of nesfatin-1 in the regulation of the reproductive axis in male rats.

Extracellular ATP a New Player in Cancer Metabolism: NSCLC Cells Internalize ATP In Vitro and In Vivo Using Multiple Endocytic Mechanisms.

Intratumoral extracellular ATP concentrations are 1000 times higher than those in normal tissues of the same cell origin. However, whether or not cancer cells use the abundant extracellular ATP was unknown until we recently reported that cancer cells internalize ATP. The internalized ATP was found to substantially increase intracellular ATP concentration and promote cell proliferation and drug resistance in cancer cells. Here, using a nonhydrolyzable fluorescent ATP (NHF-ATP), radioactive and regular ATP, coupled with high and low molecular weight dextrans as endocytosis tracers and fluorescence microscopy and ATP assays, cultured human NSCLC A549 and H1299 cells as well as A549 tumor xenografts were found to internalize extracellular ATP at concentrations within the reported intratumoral extracellular ATP concentration range. In addition to macropinocytosis, both clathrin- and caveolae-mediated endocytosis significantly contribute to the ATP internalization, which led to an approximately 30% (within 45 minutes) or more than 50% (within 4 hours) increase in intracellular ATP levels after ATP incubation. This increase could not be accounted for by either purinergic receptor signaling or increased intracellular ATP synthesis rates in the ATP-treated cancer cells. These new findings significantly deepen our understanding of the Warburg effect by shedding light on how cancer cells in tumors, which are heterogeneous for oxygen and nutrition supplies, take up extracellular ATP and use the internalized ATP to perform multiple previously unrecognized functions of biological importance. They strongly suggest the existence of ATP sharing among cancer and stromal cells in tumors and simultaneously identify multiple new anticancer targets. IMPLICATIONS: Extracellular ATP is taken up by human lung cancer cells and tumors via macropinocytosis and other endocytic processes to supplement their extra energy needs for cancer growth, survival, and drug resistance, thus providing novel targets for future cancer therapy. Mol Cancer Res; 14(11); 1087-96. ©2016 AACR.

Effect of estrogen on the expression of GnRH and kisspeptin in the hypothalamus of rats during puberty.

The aim of this study was to assess whether changes in kisspeptin and GnRH levels could be attributed to sex steroids at puberty onset. We used the ovariectomy (OVX) model in rats treated with 17ß-estradiol (E2; OVX + E2), or oil (OVX + oil), and in intact rats treated with E2 (intact + E2) or oil only (intact + oil) to determine gene expression changes of Kiss1 and Gnrh1 in the hypothalamus and protein expression of kisspeptin and GnRH in the different areas of the hypothalamus. In the intact + E2 and OVX + E2 rats on the day of the onset of puberty, GnRH-immunoreactive (ir) cell numbers decreased (P < 0.05) in the arcuate nucleus but were increased in the preoptic area; Kisspeptin-ir cells increased (P < 0.05) in the arcuate nucleus, periventricular nucleus, and preoptic area; no difference (P > 0.05) was found in the paraventricularis nucleus for GnRH-ir or kisspeptin-ir cells. Additionally, levels of Kiss1 and Gnrh1 messenger RNA in the hypothalamus were significantly higher (P < 0.05) in the OVX + E2 or intact + E2 rats than in the OVX + oil or intact + oil animals, respectively. In the OVX + oil rats, OVX significantly increased (P < 0.05) levels of Gnrh1 and Kiss1 messenger RNA and the expression of GnRH and kisspeptin in the hypothalamus compared to intact + oil animals. These results suggest that kisspeptin and GnRH play major roles in modulating the activity of estrogen circuits at the onset of puberty.

Effects of GnRH immunization on the reproductive axis and thymulin.

The bidirectional regulation of thymulin in the reproductive-endocrine function of the hypothalamic-pituitary-gonadal (HPG) axis of rats immunized against GnRH remains largely unclear. We explored the alterations in hormones in the HPG axis in immunized rats to dissect the repressive effect of immunization on thymulin, and to clarify the interrelation of reproductive hormones and thymulin in vivo. The results showed that, in the first 2 weeks of booster immunization, thymulin was repressed when reproductive hormones were severely reduced. The self-feedback regulation of thymulin was then stimulated in later immune stages: the rising circulating thymulin upregulated LH and FSH, including GnRH in the hypothalamus, although the levels of those hormones were still significantly lower than in the control groups. In astrocytes, thymulin produced a feedback effect in regulated GnRH neurons. However, in the arcuate nucleus (Arc) and the median eminence (ME), the mediator of astrocytes and other glial cells were also directly affected by reproductive hormones. Thus, in immunized rats, the expression of glial fibrillary acidic protein was distinctly stimulated in the Arc and ME. This study demonstrated that thymulin was downregulated by immunization against GnRH in early stage. Subsequently, the self-feedback regulation was provoked by low circulating thymulin. Thereafter, rising thymulin levels promoted pituitary gonadotropins levels, while acting directly on GnRH neurons, which was mediated by astrocytes in a region-dependent manner in the hypothalamus.

Variation in yield gap induced by nitrogen, phosphorus and potassium fertilizer in North China Plain.

A field experiment was conducted under a wheat-maize rotation system from 1990 to 2006 in North China Plain (NCP) to determine the effects of N, P and K on yield and yield gap. There were five treatments: NPK, PK, NK, NP and a control. Average wheat and maize yields were the highest in the NPK treatment, followed by those in the NP plots among all treatments. For wheat and maize yield, a significant increasing trend over time was found in the NPK-treated plots and a decreasing trend in the NK-treated plots. In the absence of N or P, wheat and maize yields were significantly lower than those in the NPK treatment. For both crops, the increasing rate of the yield gap was the highest in the P omission plots, i.e., 189.1 kg ha(-1) yr(-1) for wheat and 560.6 kg ha(-1) yr(-1) for maize. The cumulative omission of P fertilizer induced a deficit in the soil available N and extractable P concentrations for maize. The P fertilizer was more pivotal in long-term wheat and maize growth and soil fertility conservation in NCP, although the N fertilizer input was important for both crops growth. The crop response to K fertilizers was much lower than that to N or P fertilizers, but for maize, the cumulative omission of K fertilizer decreased the yield by 26% and increased the yield gap at a rate of 322.7 kg ha(-1) yr(-1). The soil indigenous K supply was not sufficiently high to meet maize K requirement over a long period. The proper application of K fertilizers is necessary for maize production in the region. Thus, the appropriate application of N and P fertilizers for the growth of both crops, while regularly combining K fertilizers for maize growth, is absolutely necessary for sustainable crop production in the NCP.

Up-regulation of the expression of cocaine and amphetamine-regulated transcript peptide by electroacupuncture in the arcuate nucleus of diet-induced obese rats.

It was reported that acupuncture or electro-acupuncture (EA) is effective in reducing the body weight for obese patients, although the mechanisms remain obscure. In a previous study, we have found that rats fed with high-fat (HIF) diet developed diet-induced obesity (DIO) with a concomitant decrease in the hypothalamic content of the cocaine and amphetamine-regulated transcript (CART) peptide, a peptide with anorexiogenic effect. To assess the central effect of EA on DIO rat, we revealed that EA up-regulated the expression of CART peptide in the arcuate nucleus (ARC) of the DIO rats. After feeding with HIF diet for 14 weeks, the DIO rats received EA stimulation three times per week for 4 weeks. The expression of CART peptide in ARC was measured using immunohistochemistry. The plasma ACTH was measured with ELISA. EA caused a reduction of both body weight and energy intake in DIO rats and increased the expression of CART peptide in ARC. The plasma ACTH was increased in response to restraint stress, but EA produced no further increase in ACTH levels. The results suggest that EA can up-regulate the expression of CART peptide to approach normal level, resulting in an inhibition of food intake and a reduction of body weight in DIO rats.