Insulin And Ghrelin Expression In Rats With Intrauterine Growth Retardation

BackgroundRecently, many medical studies focus on metabolic syndrome (MS), which includes glucose intolerance, dyslipidemia, obesity, and hypertension. The association between the perturbation of the early nutritional environment and the major risk factors (hypertension, insulin resistance, and obesity) for cardiovascular disease, diabetes, and the metabolic syndrome in adult life has been identified in different populations or countries by a worldwide series of epidemiological studies. Researchers tried to clarify the mechanism underlying the relation between birth weight and adult diseases. During the past decade there have been a number of mechanistic frameworks proposed to explain the biological basis of the associations observed between birth weight and health outcomes in the epidemiological studies. The "fetal" or "early" origins of adult disease hypothesis was originally put forward by David Barker and colleagues and stated that environmental factors, particularlynutrition, act in early life to program the risks for adverse health outcomes in adult life. It is also clear from experimental studies that a range of molecular, cellular, metabolic, neuroendocrine, and physiological adaptations to changes in the early nutritional environment result in a permanent alteration of the developmental pattern of cellular proliferation and differentiation in key tissue and organ systems that result in pathological consequences in adult life. The hypotheses such as "nutritional programming", "metabolic programming" etc. had been coined successively.Insulin resistance and/or β cell dysfunction contribute to the pathophysiology of low birth weight leading to the risk for MS in adults. The typical endocrine cells in pancreatic islet include α cells, β cells, δ cells and PP cells, which secret the known pancreatic hormones (insulin, glucagon, somatostatin, or pancreatic polypeptide). Ghrelin, a novel peptide of 28 amino acids, was recently isolated from rat stomach as ligand of the growth hormone secretagogue receptor (GHS-R). Ghrelin has also been detected in different tissues and organs. In 2002, Wierup et al. identified that ghrelin is expressed in a quite prominent endocrine cell population in human fetal pancreas and ghrelin expression in the pancreas precedes by far that in the stomach. Pancreatic ghrelin cell number remains in adult islets. Ghrelin is not co-expressed with any known islet hormone, and the ghrelin cells may therefore constitute a new islet cell type. Pancreatic ghrelin cells were numerous from midgestation to early postnatal life (10% of all endocrine cells). The ghrelin cells were few, occasionly seen in adults as single cells at the islet periphery. GHS-R was reported to express in the β cells and β cell lines of mouse and rat pancreatic islets throughout development. GHS-R was also identified in the rat a cells. This may suggest an autocrine or paracrine role of islet ghrelin. Mice lacking Nkx2.2 and Pax6 have relatively normal sized islets, but a large number of cells within the mutant islet fail to produce any of the four major islet hormones. Nkx2.2 or and Pax6 mutant endocrine cells have been replaced by cellsthat produce ghrelin. The expansion of ghrelin-producing cells is at the expense of β cells.The mass of endocrine tissue in the perinatal pancreas is dependent on three processes: neogenesis of the endocrine cells from the pancreatic duct epithelia, proliferation of the cells which are committed to endocrine differentiation, and apoptosis of those endocrine cells in the developing islets. During "critical windows period", Nutritional perturbations may produce long-term consequences for pancreatic structure and function.According to above background, the aims of this study were as follows:1. To analyze the relationshp between ghrelin positive cells and α cells , β cells.2. To clarify if intrauterine malnutrition alters ghrelin expression and the distribution of ghrelin positive cells in pancreatic islet and if the alteration of ghrelin positive cells is related to the change of β cells.3. To investigate the characterization of pancreatic islet ghrelin in low birth weight rats by different diet interventions and to identify the role of ghrelin in the associations between low birth weight and insulin resistance.Part One. The effects of intrauterine nutrition restriction on pancreas ghrelin and insulin expression in neonatal ratsObjectives1. To investigate the changes in plasma ghrelin, insulin and glucose concentrations in intrauterine growth restricted neonatal rats2. To compare the variation of ghrelin expression in pancreas in response to intrauterine malnutrition in neonatal rats.Subjects and MethodsAnimal modelRats at day 2 of the pregnancy were randomly divided into 2 groups: ad libitum (nourished; NR) and undernourished rats (UR). Rats in the NR group were fed a standard commercial rat diet; Rats in the UR group were fed the same diet at 50% of the NR intake, as determined by the amount of food consumed by the control group, from d 2 of pregnancy until parturition. The offspring of NR were defined as normal birth weight group (NBW n=79); the pups of UR were defined as low birth weight group (LBW n=74). Laboratory studiesPlasma glucose, ghrelin and serum insulin of both dams and their pups were analyzed at the first day after birth. Entire pancreas were collected for determination of ghrelin and insulin mRNA and quantification of pancreas ghrelin and insulin. Immunohistochemical double-staining and confocal microscopy was performed on rat pancreas.Glucose and hormone determinations: Ghrelin and insulin immunoreactivities were determined by RIA. Glucose was determined by the glucose oxidase method.Reverse transcriptase-polymerase chain reaction (RT-PCR) of ghrelin and insulin. Ghrelin and insulin mRNAs were estimated by RT-PCR. Total RNA was isolated by using Trizol. The total RNA was subjected to reverse transcription and the resulting DNA was amplified with sequence-specific primers and Taq DNA polymerase. Optical density of the bands was analyzed. β-actin was used as reference.Quantification of pancreas ghrelin content and insulin secretion Pancreas ghrelin was extracted as described by Toshinai et al. with slight modifications. Inorder to determine insulin secretion, islets were isolated by collagenase digestion and incubated for 90 min in the presence of 16.7 mmol/L glucose. The insulin was measured by RIA in the medium at the end of the incubation period.Immunofluorescence double-staining, DAPI staining and confocal microscopy Ghrelin and insulin or glucagon double fluorescence immunostaining was performed on formalin-fixed and paraffin-embedded rat pancreas. A standard manual immunofluorescence procedure was used with antigen retrieval procedure. First, the sections were incubated with a rabbit anti-rat ghrelin Subsequently, sections were incubated with Rhodamine B -labeled serum goat anti-rabbit antibody. Next, in order to demonstrate simultaneously the presence of insulin-like immunoreactivity or glucagon-like immunoreactivity, sections were incubated with a monoclonal mouse anti-rat insulin antibody or a monoclonal mouse anti- rat glucagon antibody, followed by incubation with serum goat anti-mouse antibody labeled with fluorescein-5-isothiocyanate (FITC). Control experiments were included in serial sections by either omitting the primary antibody. The slides were then counterstained with the nuclear stain 4'-6-Diamidino-2-phenylindole (DAPI) to detect the number of islet cells. Double immunolabeling was detected by a Zeiss LSM510 confocal laser-scanning microscope. The final tricolor images were compiled from overlaid images of the blue (nuclei), green (insulin or glucagon), and red (ghrelin) images. Point counting for double-positive cells was used for a quantitative analysis of the number of islet cells containing insulin or ghrelin at various sections. Five pancreases were subjected to quantitative analysis in each group.ResultsEffect of maternal undemutrition on plasma ghrelin and insulin concentrationsBody weight of both dams and fetuses was significantly affected by feeding status (p< 0.001). Plasma total ghrelin concentrations were significantly affected by feeding state (p< 0.001). After parturition, fasting plasma ghrelin concentrations in UR group [1382 (1287-1513) pg/ml] was higher than that of NR group [1072 (974-1205) pg/ml]. At the first day after birth, pups plasma ghrelin concentrations in the LBW group [2176 (2031-2384) pg/ml] were significantly lower than that of NBW group [2493 (2311-2675) pg/ml]. Plasma insulin concentrations were significantly affected by nutrition state (p< 0.001). Undemutrition caused a decrease in plasma insulin concentrations in both UR dams and LBW pups. Plasma glucose concentrations were also significantly affected by maternal nutrition state (p < 0.001).Effect of intrauterine undemutrition on pups pancreas ghrelin and insulin gene expressionGhrelin mRNA and concentrations of total ghrelin in the pancreas were significantly affected by intrauterine nutrition state. Pancreas ghrelin/β-actin ratio [372% (307-513%)] in the LBW group was significantly higher compared with that in the NBW group [124% (57-198%)]. Pancreas total ghrelin content was 714 (632-759) ng/g tissues in the LBW group compared with 479 (381-732) ng/g tissues in the NBW group, there was difference in pancreas total ghrelin content between the two groups (p<0.01). Pancreas insulin/β-actin ratio (expressed as percentage) was 112% (67-194%) in the LBW group compared with 183% (157-302%) in the NBW group, respectively. The results show that insulin content was 782.1 (522-986)pmol/islet-90min and 2448.9 (1987-2954) pmol/islet-90min in LBW group and NBW group, respectively. Pancreas insulin concentrations of newborn rats were significantly affected by intrauterine nutrition (p = 0.007). Ghrelin expression in pancreatic isletsA strong insulin immunoreactivity was showed at the center of NBW pancreatic islets, most of ghrelin-immunoreactive cells distributed at the periphery of the islets. In pancreatic islet of LBW, most of insulin and ghrelin were colocalized in the central of islets; majority of insulin-producing cells (or β cells) co-expressed ghrelin. Ghrelin had a preferential central distribution within the endocrine islet of LBW group. The percent of ghrelin~+ insulin~+cells in LBW group was 33.7% (95%CI: 16.2%-41.1%)]. Many glucagon~+ cells coexpressed ghrelin at the periphery of the islets of NBW, the percent of ghrelin~+ glucagon~+ cells was 5.3% (95%CI: 3.9%-9.7%) in NBW group. Two populations of cells in the islet of LBW group were detectable: cells uniquely expressing glucagon at the periphery of the islets or cells uniquely expressing ghrelin at the center of the islets. The percent of ghrelin~+ cells and insulin~+ cells was 83.1% (95%CI: 76.2%-90.1%) in the islets of LBW group and 90.1% (95%CI: 85.7%-95.1%) in NBW group respectively. The percent of ghrelin~+ cells in the islets of LBW [48.7% (95%CI: 33.2%-56.7%)] was significantly higher than that of NBW group [15.3% (95%CI: 7.2%-20.1%)].Conclusions1. Intrauterine undernutrition affects the birth weight, plasma insulin and ghrelin levels.2. Intrauterine undernutrition affects islet ghrelin expression and ghrelin-expressing cell distribution.Part Two. The age-dependent alteration of insulin and ghrelin expression in the pancreatic islets of intrauterine growth restricted rats following different dietinterventionObjectives1. To analyze the age-dependent alteration of insulin and ghrelin expression in the pancreatic islets of IUGR rats during development.2. To investigate the effects of different diets on the pathophysiology process of intrauterine growth retardation rats and the risk for MS and to identify the role of ghrelin in the associations between low birth weight and insulin resistance.Subjects and MethodsAnimal Model and GroupNeonatal SD rats were divided into 6 groups as follows. All were raised with their dams in the same cage till weaning. After parturition, the dams were fed different diets ad libitum. After weaning, the pups were fed the same diets as their dams respectively. Rats were studied at day 1 (after birth), 3, 7, and 10 and at 2 week, 3 week, 4 week, 8 week and 12 week.1. normal birth weight-normal diet group, N-N- group2. normal birth weight-high lipid and high energy group, N-H- group3. normal birth weight-high protein group, N-P- group4. low birth weight-normal diet group, L-N- group5. low birth weight-high lipid and high energy group, L-H- group6. low birth weight-high protein group, L-P- groupLaboratory studiesPlasma glucose, ghrelin and serum insulin were analyzed. Entire pancreas were collected for determination of ghrelin and insulin mRNA and quantification ofpancreas ghrelin and insulin. Immunohistochemical double-staining and confocal microscopy was performed, which was the same as Part one.Results1 The weight of pup rats in different groups at different time.The weights of all the LBW groups were lower than those of matched NBW groups fed the same diets respectively from day 1 to 2 weeks(p<0.05). The 'catch up growth' phenomenon wasn't found in LBW group from day 1 to 2 weeks. Subsequently, the weight difference between LBW group and NBW group decreased. The weights of N-H group and N-P group were higher than those of N-N group at 12 weeks (p<0.05). There were no significant difference in weights among different LBW groups at the same time(p>0.05).2 The comparison of fasting glucose levelsThe fasting glucose levels of N-H group were higher than those of N-N group from the age of 4 week to 12 week after birth (p<0.05). The fasting glucose levels of L-H group were higher than L-N group from the age of 2 week to 12 week after birth (p<0.05). The fasting glucose levels of L-N group were higher than those of N-N group, there were significant difference between them (p<0.05). The fasting glucose levels of N-P group increased significantly at the age of 12 weeks, however, the fasting glucose levels of L-Pgroup didn't show increase at the age of 12 weeks.3 The alteration of plasma insulin concentration and the homeostasis model assessment-insulin resistance index (HOMA-IR)The plasma insulin levels of LBW groups were lower than those of matched NBW groups at day 1, 3 and 7 after birth (p<0.05). The plasma insulin levels became close among all the groups from day 10 to 8 weeks, there were no difference in the plasma insulin levels among group during this period. The plasma insulin levels of L-H group and N-H group were significantly higher than those of L-N group and N-Ngroup at 12 week respectively (p<0.05).The HOMA-IR of LBW groups was higher than those of matched NBW groups fed the same diets respectively at day 1, 3 and 7 (p<0.05) . The HOMA-IR increased gradually by a time-dependent way in the L-H and N-H group. At 12 week, the HOMA-IR of L-N group was significantly higher than those of N-N group(p< 0.05), and the HOMA-IR of L-H group was significantly higher than those of N-H group(p<0.05). However, the HOMA-IR of L-P group was significantly lower than those of N-P group(p<0.05). Compared with the values of day 1 after birth, the HOMA-IR of N-H group, N-P group and all the LBW subgroups increased significantly (p<0.05) at 12 weeks.4 Plasma ghrelin concentrationsPlasma ghrelin concentrations of all the groups decreased in an age-dependent manner. At day 1, 3 and 7, plasma ghrelin concentrations of LBW groups were lower than those of NBW groups (p<0.05). Though the plasma ghrelin concentrations of LBW groups were a little higher than those of NBW groups at 4, 8 and 12 weeks, there were no significant differences between those diet-matched groups(p>0.05).5 The insulin secretion function of pancreatic islets in different groupsThe islet insulin secretion levels of LBW groups were lower than those of diet-matched NBW groups at any matched time, though there were significant differences only between L-H group and N-H group at different time (p<0.05). The islet insulin secretion levels of N-H group and N-P group were lower than N-N group at 4, 8 and 12 weeks (p<0.05). The islet insulin secretion levels of L-H group were lower than L-N group (p<0.05); However, insulin secretion levels of L-P group were higher than L-N group (p<0.05).6 The pancreatic ghrelin contentsPancreatic ghrelin contents of all the groups decreased in an age-dependentmanner. The pancreatic ghrelin contents of LBW groups were higher than those of diet-matched NBW groups at any matched time (p<0.05). Pancreatic ghrelin contents of N-H group and N-P group decreased more rapidly than N-N group from the 2nd week to the 4th week (p<0.05). Though pancreatic ghrelin contents of L-H group were lower than L-N group at 8th week and 12th week after birth, pancreatic ghrelin contents of L-P group were higher than L-N group at the 8th week and the 12th week after birth, there were no differences between them (p>0.05).7 Pancreatic insulin mRNA expressionThe pancreatic insulin mRNA expression of different LBW groups was lower than those of diet-matched NBW groups at any matched time (p<0.05). There were no differences in pancreatic insulin mRNA expression among NBW subgroups at different age (p>0.05). Pancreatic insulin mRNA expression of L-H group were lower than L-N group at 8th week and 12th week after birth (p<0.05). Pancreatic insulin mRNA expression of L-P group were higher than L-N group at the 12th week, there were no differences between them (p>0.05).8 Pancreatic ghrelin mRNA expressionPancreatic ghrelin mRNA expression of all the groups decreased in an age-dependent manner. At day 1, 3 and 7, Pancreatic ghrelin mRNA expression of LBW group was higher than that of NBW group (p<0.05). There were no differences in pancreatic ghrelin mRNA expression among NBW subgroups at different age (p>0.05). Pancreatic ghrelin mRNA expression of L-P group were higher than L-N group at the 4th, 8th and 12th week after birth, pancreatic ghrelin mRNA expression of L-H group were lower than L-N group, there were no differences between them (p>0.05).9 The changes of insulin-positive (insulin~+) cells and ghrelin-positive (ghrelin~+) cells in pancreatic islets during developmentThe percent of insulin~+ cells in pancreatic islets decreased gradually in an age-dependent manner. Though the percent of insulin~+ cells in LBW groups was higher than the same diet NBW group, there was no significant difference between those matched two groups at different time (p>0.05). There were no difference in the percent of insulin~+ cells among the three NBW subgroups at different time (p> 0.05).The same results can be found in LBW rats. There were no difference in the percent of insulin~+ cells among the three LBW subgroups at different time (p>0.05).The percent of ghrelin~+ cells in pancreatic islets also decreased gradually in an age-dependent manner, and it descended more rapidly from day 1 to day 10 than other duration (p<0.05). The percent of ghrelin~+ cells in LBW groups was higher than those of LBW groups at the same age from day 1 to 8th weeks. There were no significant differences in the percent of ghrelin~+ cells among three NBW subgroups at the same age(p>0.05). Though the percent of ghrelin~+ cells in L-P group was higher than L-N group at the same age from day 7 to week 4, there were no differences between them (p>0.05).The distributions of gherlin~+ cells in pancreatic islets of LBW groups were different from those of NBW groups. In NBW rats, ghrelin~+ cells distributed at the periphery of the islets, the number of ghrelin~+ cells gradually decreased with the growth of NBW rats. No ghrelin~+ cells can be detected in some pancreatic islets of NBW rats at the age of 12 weeks. In pancreatic islet of LBW rats, most of ghrelin~+ cells were located in the central of islet, A few ghrelin~+ cells also can be detected at the periphery of the islets. With the growth and development of LBW rats, ghrelin~+ cells in the central of islet decreased and disappeared more quickly. Ghrelin~+ cells at the periphery of the islets also gradually decreased, however, at the age of 12 weeks, ghrelin~+ cells still can be detected at the periphery of each islet in LBW rats.Conclusions1. The effects of IUGR on weight, plasma ghrelin levels and insulin secretion of pups ratspersisted after birth.2. High lipid and high energy diets can induce IR in IUGR rats, aggrevate insulin secretion dysfunction, which contributes to the associations between low birth weight and MS.3. High protein diets in IUGR rats can delay or decrease the incidence of hyperglycemia. The ceasation of ghrelin expression in the pancreatic islets was delayed in IUGR rats, which indicates that ghrelin may be a protective factor in preventing IUGR rats from getting T2DM.