Effects Of Zinc On The Growth Performance Of Rats And The Approach To Mechanism For Zinc And CGRP Regulation Of Food Intake

Zinc (Zn) is an essential trace element required for animals. Although many studies about biological functions of zinc have been carried out in recent years, the molecular mechanism for zinc nutrition is still undefined till now, in particular, the pathway for zinc regulation of food intake. The present study was conducted to investigate effects of three Zn levels, namely deficiency, adequacy, and overdose, on the food intake, growth and development, metabolism and gene expression profiling in pituitary of SD rats; meanwhile, basing on the results which Zn deficiency largely decreased food intake and increased the mRNA level of calcitonin gene-related peptide (CGRP), the mechanism for Zn and CGRP regulation of food intake was further explored in the subsequent study. The main contents and results were presented as follows:1 Effects of zinc on the food intake, growth and development, metabolism and gene expression profiling in pituitary of SD ratsTwo hundreds and forty male Harlan Sprague-Dawley outbred weaning rats (25-day old and weighing 95±5 g) were used in this study. After a 3-d period of acclimation, animals were randomly divided into three groups. The first group was fed a Zn-deficient diet (ZD; 3.15 mg/kg), the second group was fed a Zn-adequate diet (ZA; 35.94 mg/kg, control) and the third group was fed a Zn-overdose diet (ZO; 347.50 mg/kg). Sixty rats in ZD and ZO group, respectively, and 120 rats in ZA group were fed for 6 weeks in barrier system. Body weight and food intake were recorded weekly. The results showed that rats fed the Zn-deficient diet showed typical symptoms such as lethargy and lower activity, poor appetite, sparse and coarse hair, unhairing and leanness; whereas, rats fed the Zn-overdose diet maintained good phenotype and ingestive behavior similar to rats fed the Zn-adequate diet. Compared with ZA group, Zn deficiency significantly (P<0.05) reduced food intake, body and organ weight, Zn levels, alkaline phosphatase (ALP), copper zinc superoxide dismutase (Cu-Zn SOD) and amylase activities, and increased metallothlonein (MT) contents, but had no effect (P>0.05) on maleic dialdehyde (MDA) levels, total-proteolytic enzyme and lipase activities; whereas, Zn overdose was marked (P<0.05) by higher body and organ weight, Zn levels, ALP activity and MT contents, and lower lipase activity, but was without effect (P>0.05) on the other parameters mentioned above.The results derived from analyzing the changed genes in expression profiling by means of clustering and functioning showed that Zn deficiency or Zn overdose affected the expression patterns of pituitary genes which were involved in a variety of biological functions, including DNA replication and transcription, nucleic acids processing and repair, nutrients metabolism, feeding, signaling, ions transport, stress and immunity, antioxidant and so on. As for the gene expression of the appetite-related neuropeptides, Zn deficiency downregulated neuropeptide Y (NPY) mRNA levels, but upregulated cholecystokinin (CCK) and CGRP mRNA levels; while Zn overdose upregulated melanin-concentrating hormone (MCH) and ghrelin mRNA levels.2 The approach to mechanism for Zn and CGRP regulation of food intakeThe experiment was done in two parts. First, the adaptive response of SD rats to Zn and CGRP intraperitoneal injection was studied using lithium chloride (LiCl) as a positive control, in order to determine the proper injection dose of Zn and CGRP. In the second part, basing on the establishment of two Zn models by feeding SD rats with the Zn-deficient (3.30 mg/kg) and Zn-adequate (38.89 mg/kg) diets, the 4x2x2 trifactorial replicated experimental design was used to investigate the effects of Zn injection (A factor; with four concentrations of Zn,0,0.5,1.0 and 2.0μg/g), CGRP injection (B factor; with two concentrations of CGRP,0 and 0.05μg/g) and Zn models (C factor; including Zn deficiency and Zn adequacy) on the food intake of SD rats, gastrointestinal digestive functions, biochemical metabolism, neuroendocrine system and feeding-related signaling and gene expressions of neuropeptides, the corresponding correlation analysis was also simultaneously performed in the present study. The main results were listed as follows:2.1 The adaptive response of SD rats to Zn and CGRP intraperitoneal injectionThe rat injected with 150μg/g of LiCl showed a significant less consumption of 0.1% saccharin solution than purified water (28.55%, P<0.05), however, there were no remarkable (P>0.05) differences in the consumption of purified water and saccharin solution of rats injected with 1.0μg/g and 2.0μg/g of Zn, and 0.05μg/g of CGRP, which was similar to that of rats injected with saline. The results demonstrated that Zn and CGRP injection under the limited dosage did not induce untoward effect or malaise, thereby the probability of Zn or CGRP influenced food intake due to causing malaise was excluded.2.2 Establishment of Zn deficiency and Zn adequacy models in SD ratsThe results showed that rats fed the Zn-deficient diet revealed symptoms typical of lethargy and lower movements, poor appetite, susceptible to environmental variables, disorder and reluster of hair, and leanness; whereas, rats fed the Zn-adequate diet exhibited normal mental state and ingestive behavior. Serum, femur and skeletal muscles Zn levels of rats fed the Zn-deficient diet were lower than that of rats fed the Zn-adequate diet by 26.58%(P<0.01),27.32%(P<0.01) and 24.22%(P<0.05); respectively. The findings indicated that Zn deficiency and Zn adequacy models in SD rats were estabolished successfully.2.3 Effects of Zn injectionCompared with saline-injected control group, Zn injection with the dose of 0.5,1.0 and 2.0μg/g had no effect (P>0.05) on food intake, duodenum hydrolases including total-proteolytic enzyme, amylase and lipase activities, and serum glucose (Glu) level, but enhanced (P<0.05) pepsin activity, and decreased (P<0.05) serum triglyceride (TG) level. Compared with the control group, the changes in the levels of CGRP, NPY, leptin, gastrin, insulin, glucagon, thyroid hormone (T3 and T4) were not observed (P>0.05) in the group injected with 0.5μg/g Zn; the NPY level was increased (P<0.05) in both the group injected with 1.0μg/g Zn and 2.0μg/g Zn, but the variations in insulin level were just the contrary; the levels of CGRP and gastrin were elevated (P<0.05), but the glucagon level was reduced (P<0.05) in the group injected with 2.0μg/g Zn. The MCH mRNA level was increased (P<0.05) in three Zn-injected groups, and NPY mRNA level was also enhanced (P<0.05) in the 2.0μg/g Zn-injected group as compared to saline-injected group, while the levels of CGRP and CCK mRNA were unaffected (P>0.05) by Zn injection. The administration of 2.0μg/g Zn was without effect (P>0.05) on cAMP levels in spite of inhibitory effect of the injection of two lower dose (0.5 and 1.0μg/g) Zn.2.4 Effects of CGRP injectionCompared with saline-injected group, injection of 0.05μg/g of CGRP suppressed (P<0.05) food intake. CGRP (mRNA and polypeptide), glucagon, cAMP and CCK mRNA levels were all elevated (P<0.05), but pepsin and amylase activities, and Glu, gastrin, insulin, NPY (mRNA and polypeptide) and MCH mRNA levels were all declined (P<0.05) after treatment with 0.05μg/g CGRP in comparison to control (saline-treated) group. There were no significant (P>0.05) differences in the activities of total-proteolytic enzyme and lipase, and the levels of TG, leptin, T3 and T4.2.5 Effects of Zn modelCompared with Zn adequacy, Zn deficiency depressed (P<0.05) food intake. Zn deficiency also caused lower (P<0.05) activities of pepsin and amylase, and levels of Glu, TG, leptin, gastrin, insulin, T3 and T4, but resulted in higher CGRP (mRNA and polypeptide), NPY (mRNA and ploypeptide), CCK mRNA and cAMP levels. The other parameters mentioned above were unchanged (P>0.05) in the Zn-deficient group in contrast to the Zn-adequate group.2.6 Correlation analysisThere was a significant positive correlation between food intake and the parameters including MCH mRNA, T3, T4, pepsin and amylase, their correlation coefficients (r) were 0.467 (P<0.01),0.439 (P<0.01),0.399 (P<0.01),0.320 (P<0.01) and 0.266 (P<0.05), respectively; a significant negative correlation existed between food intake and CGRP mRNA (r=-0.383, P<0.01), CGRP polypeptide (r=-0.531, P<0.01), cAMP (r=-0.309, P<0.05), glucagon (r=-0.331, P<0.05) and CCK mRNA (r=-0.471, P<0.01) levels; respectively. In addition, there was a remarkable positive or negative correlation among some parameters. The positive relationships were CGRP mRNA vs CCK mRNA (r= 0.368, P<0.01), CGRP mRNA vs cAMP (r= 0.304, P<0.05), CGRP polypeptide vs CCK mRNA (r= 0.365, P<0.05), CGRP polypeptide vs cAMP (r= 0.377, P<0.05), CGRP polypeptide vs glucagon (r= 0.563, P<0.01), gastrin vs pepsin (r= 0.392, P<0.01), gastrin vs amylase (r= 0.501, P<0.01), insulin vs leptin (r= 0.292, P<0.05), insulin vs TG (r= 0.333, P<0.05) and leptin vs TG (r= 0.289, P<0.05). The negative relationships included CGRP polypeptide vs insulin (r=-0.362, P<0.05), CGRP polypeptide vs MCH mRNA (r=-0.500, P<0.01), CGRP polypeptide vs gastrin (r=-0.290, P<0.05), CGRP polypeptide vs amylase (r=-0.411, P<0.01), cAMP vs T3 (r=-0.387, P<0.01), cAMP vs T4 (r=-0.333, P<0.05) and glucagon vs MCH mRNA (r=-0.597, P<0.01).