Expression Of Ghrelin And Its Relationship With Insulin And Leptin In Diabetic Rats

BackgroundAs a growing number of people suffer from metabolic disorders, such as obesity, diabetes mellitus and so on, understanding the mechanisms by which various hormones and neurotransmitters have influence on energy balance has been a subject of intensive research.Ghrelin is a 28-amino acid peptide, which is both an endogenous ligand for the GH secretagogue receptor and an orexigenic hormone implicated in the control of food intake and energy balance. Ghrelin is produced by the endocrine A-like cells, which are mainly found in the oxyntic mucosa in the stomach, to a lesser extent in the gastric antrum and, sparsely, in the small intestine. Ghrelin has also been detected in the hypothalamus suggesting its involvement in energy homeostasis and GH release. The secretion of ghrelin by the stomach depends largely on the nutritional state. Ghrelin levels show preprandial increases and postprandial decreases. Among several mechanisms forwarded to explain nutrient-induced inhibition of ghrelin secretion is the postprandial release of the pancreatic hormone insulin. This hypothesis is supported by the inverse temporal profiles of plasma ghrelin and insulin levels both before and after meals and by evidence that sustained, pharmacological elevations of plasma insulin lower plasma ghrelin concentrations. Findings on this point are controversial, however, and the physiologic role of insulin in the control of ghrelin release remains uncertain. Leptin, a 167-amino acid product of ob gene, is a peripheral adiposity signal for the central regulation of food intake and energy homeostasis. Leptin is maily produced by adipocytes, but also produced in small amounts in other human tissues such as the stomach, mammary epithelium, placenta and heart. Most interestingly, leptin functions as a feedback mechanism that signals to key regulatory centers in the brain to inhibit food intake and to regulate bodyweight and energy homeostasis. Notably, leptin has also been suggested to have influence on circulating ghrelin levels. It has been hypothesized that the satiety-inducing effects of leptin include the suppression of ghrelin secretion. Indeed, the effects of leptin on energy homeostasis are opposite (although not complementary) to those of ghrelin; leptin induces weight loss by suppression of food intake, whereas ghrelin functions as an appetite-stimulatory signal. Moreover, leptin has been shown to be an upstream regulator of ghrelin in rodents. However, several studies in humans have produced conflicting results. A recent study showed that leptin administration- to healthy volunteers does not regulate ghrelin levels over several hours to a few days .It can be seen that both ghrelin and leptin are involved in energy balance of the body, which may act on carbohydrate metabolisms and insulin secretion or activity. However, researches on the expression of ghrelin in diabetes mellitus and its interactions with insulin or leptin are rare, so we examined the plasma ghrelin levels and mRNA expressions in the stomach both in type 2 diabetic rats and STZ-induced insulin-deficient diabetic rats.Part 1. EFFECTS OF PIOGLITAZONE ON GHRELINEXPRESSION OF TYPE 2 DIABETIC RATSObjective Wastar rats fed with high fat diet and treated by low dose streptozotocin (STZ) were used as diabetic model in this research. Blood levels of ghrelin, leptin and insulin, the mRNA and protein expressions of ghrelin were analyzed in order to reveal the relationship of ghrelin with leptin, insulin and other related factors and to determine the effect of Pioglitazone on them. Methods Fourty wistar rats were divided into three groups: normal control(NC), diabetes group(DM) and pioglitazone treating group(DM-PIO). Diabetic rats were fed with high fat diet for 8 weeks and then treated with low dose of STZ(30mg/kg) by intraperitoneal injection to establish type 2 diabetic rat models. Afterwards, rats in DM-PIO group were treated with pioglitazone(20mg·kg^-1·d^-1) by intragastric administration and all the animals were sacrificed 8 weeks later. Under general injection anesthesia, the rats were euthanized by decapitation. Blood samples were collected for measurement of blood glucose, lipids, insulin, leptin and ghrelin. Specimens from the stomach were studied by immunocytochemistry for ghrelin protein and RT-PCR for ghrelin mRNA expression. Data were presented as mean±SEM and analysed using the Statistical Package of the SPSS 11.0. Results1. The body weight, serum fasting TG and insulin levels and HOMA-IR were significantly higher in diabetic rats than those in NC rats after 8 wk of high fat feeding(P＜0.01, 0.05, 0.01, 0.05, respectively).2. After 2 wk of STZ injection, the FBG (P＜0.01) and serum fasting TC (P＜0.05) were also significantly increased in diabetic rats compared to the values in NC rats.3. The body weight, FFA, HOMA-IR and serum fasting leptin levels were significantly higher in DM-PIO rats than those in DM rats (P＜0.05 for each) after 8 week of treatment and the FBG, FINS, TG and TC levels in DM-PIO group were, though not significantly, tended to reduce in DM-PIO group compared with DM group.4. Both DM and DM-PIO rats had significantly reduced plasma levels of ghrelin compared to those in NC rats (P＜0.05 for each). Plasma ghrelin levels were higher in DM-PIO rats than those in DM rats though not significantly. By correlation analysis, plasma ghrelin levels were found negatively correlated with body weight (r=-0.413,P＜0.05), HOMA-IR (r=-0.549,P<0.01) and leptin (r=-0.470,P<0.05) in diabetic rats.5. The ghrelin mRNA expression levels in the stomach were significantly lower both in DM and DM-PIO rats than those in NC rats (P＜0.01 for each) . The DM-PIO rats had higher levels of ghrelin mRNA expression in the stomach than those in DM rats though not significantly.6. The positive stained granules of ghrelin protein in the gastric fundus were significantly reduced both in DM rats and DM-PIO rats compared with NC rats (P＜0.05 for each). There was no significant difference between DM and DM-PIO rats.Conclusions:1. Type 2 diabetic model in rats can be successfully induced by high fat diet and low dose STZ injection. Insulin resistance developed with the maintenance of insulin secretion and the increase of blood glucose.2. The plasma ghrelin levels and the expression of ghrelin in diabetic rats were significantly reduced compared with normal controls, which indicated that the low plasma ghrelin levels might be regarded as an indicator of type 2 diabetes.3. Plasma ghrelin levels were negatively correlated with HOMA-IR in diabetic rats. Therefore, low ghrelin levels may be involved in the development of hyperinsulinemia and insulin resistance in type 2 diabetes.4. Diabetic rats had increased leptin levels and a significant negative association of ghrelin with leptin. Ghrelin may contribute to the leptin resistance.5. Pioglitazone can reduce HOMA-IR and serum FFA and leptin levels and improve the insulin and/or leptin resistance in diabetic rats, but not change the plasma levels of ghrelin and its expression in the stomach.Part 2. EFFECT OF LEPTIN ON GHRELIN EXPRESSIONOF STZ-INDUCED DIABETIC RATSObjective This study was conducted in streptozotocin (STZ)-induced diabetic rats featuring with insulin deficiency and many pathological and biochemical changes that resemble human type 1 diabetes. The effects of recombinant leptin on the expression of ghrelin mRNA in the stomach, leptin mRNA in the adipose tissues and some other parameters such as food intake, body weight change, blood glucose, insulin,leptin and ghrelin levels were monitored to investigate the possible relations between leptin and ghrelin in STZ-induced diabetic rats.Methods Thirty wistar rats were divided into four groups: control group (NC), diabetes group (DM), diabetes group treated with leptin (DM-LEP) and DM-LEP pair-fed group (DM-PF). The food intake of the latter group matched that of the DM-LEP group to exclude the secondary effect of leptin-induced feeding suppression. All the rats except normal controls were treated with STZ (60mg/kg) by intraperitoneal injection to establish insulin-deficient diabetic models. DM-LEP rats were injected once daily for 7 days with leptin at a dose of 4mg·kg^-1·d^-1 sc. The cumulative food intake was measured once daily for 7 days, and each group was measured for changes in body weights, blood glucose, insulin, serum leptin and leptin mRNA expression in the adipose tissues, plasma ghrelin and ghrelin mRNA expression in the stomach. Data were presented as mean±SEM and analyzed using the Statistical Package of the SPSS 110. Results:1. The food intake significantly reduced in the DM-LEP group compared to that in the DM group (P<0.05). The DM-LEP group had greater body weights compared with the DM and DM-PF groups (P＜0.05 for each).2. The DM group had higher blood glucose levels than the control group (P <0.01), and treatment with leptin decreased plasma glucose levels compared with those in the DM and DM-PF group (P<0.05 for each).3. There was no significant difference in plasma insulin levels between the DM and DM-LEP group on the 3rd or 7th day after recombinant leptin injection (P>0.05).4. The serum levels of leptin were significantly lower in diabetic rats than those in normal controls. The leptin mRNA expression in the adipose tissues was lower in the DM-LEP group than that in the DM and DM-PF group (3rd day: P＜0.05 for each, 7th day: P<0.01 for each), but still significantly increased compared with NC group (P＜0.05).5. Plasma ghrelin levels increased in the DM group compared with those in the NC group, and leptin treatment decreased plasma ghrelin levels in the DM-LEP group compared with those in the DM and DM-PF group (3rd day: .P＜0.05 for each, 7th day: P＜0.01 for each). In addition, correlation analysis showed that plasma ghrelin levels were negatively correlated with insulin (r=-0.58, P<0.01)and leptin (r=-0.63, P＜0.01) .6. The expression of ghrelin mRNA in the stomach was higher in the DM group than that in the control group (P<0.01). The leptin treatment decreased the ghrelin mRNA levels relative to those in the DM group (P<0.05).Conclusions:1. The STZ-induced diabetic rats had reduced serum leptin and leptin mRNA levels in the adipose tissues and increased plasma ghrelin and ghrelin mRNA levels in the stomach.2. Leptin improved weight loss, blood glucose and food intake values in STZ-induced diabetic rats.3. At the dose of 4mg kg^-1·d^-1, the leptin action was incomplete, and thus blood glucose levels were higher in the DM-LEP group than those in the NC group.4. Leptin did not change fasting insulin levels, but increased the insulin sensitive index.5. Peripheral leptin action elevated both blood leptin levels and leptin mRNA expression in the adipose tissues.6. Peripheral leptin action reduced food intake as well as blood ghrelin and ghrelin mRNA levels. DM-LEP rats exhibited decreased plasma ghrelin concentrations and ghrelin mRNA levels compared with those in DM-PF rats, suggesting that the leptin regulation of plasma ghrelin and ghrelin mRNA levels is independent of food intake.