Effects And Mechanism Of CART On Type2 Diabetic Rats’ Hypothalamus And Islet β Cells

1. Background and Objective:Type 2 diabetes is the most common form of diabetes （accounts for 90-95%） and is primarily characterized by insulin resistance or reduced insulin sensitivity, combined with reduced insulin secretion and hyperglycemia. This metabolic disease is a growing public health problem. Improper diet which is caused by hormone adjustment disorder is the important reason for type 2 diabetes. In recent years, the cocaine- and amphetamine-related transcript peptide （CART） plays an important role on eating behavior and in vivo energy-regulating and draws more and more attention. It was suggested that CART was involved in food intake; this suggestion was based on the distribution of CART peptides in the brain, namely in regions that included the arcuate nucleus, the dorsomedial hypothalamus nucleus, the paraventricular nucleus and the nucleus accumbens, all of which have a role in the regulation of food intake. The discovery of numerous novel neuropeptides with distinct hypothalamic distribution patterns has certainly added to the complexity of the hypothalamus they have also been the key to a better understanding of how the hypothalamus integrates neural and hormonal inputs/signals into coordinated neuroendocrine, autonomic and behavioral responses. Recent studies display that CART peptide containing cell bodies in the hypothalamic nucleus were found to be surrounded by other transmitter nerve terminals, suggesting that CART plays a role in the regulation of feeding. CART peptides and their effects have been found in feeding-relevant parts of the brain, they were also demonstrated to be present in the gut and in vagal nerves （Intestinal muscle plexus, islet cells, somatostatin gastric antrum G cells, beta cells, etc）, suggesting that CART peptides have emerged as important islet regulators. CART peptides have emerged as major neurotansmitters and hormones, are widely distributed in the CNS and are involved in regulating many processes, including food intake, the maintenance of body weight, reproduction, stress, pain, reward and endocrine functions. In this study, we investigated the effects and mechanism of CART on STZ-diabetic rats’hypothalamus and isletβcells. Our findings provide a novel insight into understanding of biological function and mechanism of CART. A role for CART in type 2 diabetes pathophysiology could become new drug targets for gene therapy in type 2 diabetes.2. Methods:2.1 To induce type 2 diabetic rats and measurement of FBG、TG、TC、FFA and FINS in bloodAfter 4 weeks feeding of the high-fat diet （to induce hyperlipidemia）, fasted rats were rendered diabetic by the intraperitoneal injection of 35 mg/kg streptozotocin dissolved in citrate-phosphate buffer and control rats were injected with the same volume citrate-phosphate buffer. One week later, rats with fasting blood glucose level of above 16.7 mmol/L were considered diabetic and were used in further studies. Total cholesterol （TC）, triglyceride （TG）, free fatty acid （FFA）, serum insulin levels in blood were measured by commercial kit.2.2 Effects of hypothalamic injection of CART on food intake and dietary intervention on CART mRNA expressionKopf sterotaxic frame and the rat brain atlas of Paxions and Watson were used to identify the nucleus. Rats were allowed to recover for 7 days after surgery. The placement of the hypothalamic cannulas was verified by histological examination of the brain at the end of the study. In fasted rats, each dose CART were injected in ARC, PVN and DMH, in satiated rats, high dose CART was injected in ARC, after injection, rats were returned to cages and the food remaining 1, 2, 4, 8, 24h after injection was weighed. Quantitative real-time reverse transcription-polymerase chain reaction （QRT-PCR） was used to measure CART mRNA expression in the hypothalamus of rats after dietary intervention.2.3 Effects of hypothalamic injection of neuropeptide Y （NPY） and CART on food intake and effects of CART on hypothalamic neuropeptide releaseIn fasted rats, each concentration of NPY or CART or the combined were injected in ARC, after injection, rats were returned to cages and the food remaining 1, 2h after injection was weighed. Double fluorescence immunohistochemistry was used to observe NPY and CART expression in PVN. Static hypothalamic explant culture was used to detect NPY, AGRP,α-MSH and CRH immunoreactivity by radio immunoassay.2.4 Effects of PKA/CREB-mediated signalingUsing in situ hybridization analysis for CART mRNA expression and Western blotting analysis for CART peptide and CREB/p-CREB expression were carried out. To find CART expression in vivo in the rat ARC is regulated by PKA-mediating signaling and likely through the activation of CREB.2.5 Effects of ERK signaling pathways activated by calcium influx in the hypothalamus neurons phosphorylation by CARTFirstly, the hypothalamic neurons from 2-3 days old SD neonatal rats were primarily cultured, and then Western blotting analysis for ERK1/2/p-ERK1/2 expression was carried out. The intracellular Ca²⁺ in these cells was marked with calcium fluorescent probe of Fluo-3/AM. The fluorescent values were recorded continuously under confocal laser scanning microscope.2.6 CART regulates islet hormone secretion and is expressed in theβcells of type 2 diabetic ratsWe have examined the role of CART as a regulator of islet hormone secretion using INS-1 cells and isolated rat islets. Fluorescence immunohistochemistry, ultrathin sections and protein A-gold technique were used to detect the CART labeling in theβcells of type 2 diabetic rats. The last part was designed to assess the chornic effects of dipeptidyl peptidase-4 inhibitor Sitagliptin treatment on oral glucose tolerance test, hormone profiles, glycemic control and pancreatic insulin content in type 2 diabetic rats. These data favor a role of CART in islet function and in the pathophysiology of type 2 diabetes.3. Results:3.1 To induce type 2 diabetic rats and measurement of FBG、TG、TC、FFA and FINS in bloodFasting blood glucose, TG, TC, FFA levels in diabetic rats were all significantly higher than that of the control ones, while serum insulin level was significantly lower. These results indicated that type 2 diabetic rats with hyperlipidemia were successfully induced.3.2 Effects of hypothalamic injection of CART on food intake and dietary intervention on CART mRNA expressionIn fasted rats, injection of high dose CART into PVN of diabetic rats significantly increased feeding 2-4h after injection compared with that in the control, vehicle and low dose CART groups, other time points did not differ significantly from control levels. Injection of low and high dose CART into DMH and ARC of diabetic rats significantly increased feeding 1-2h and 2-4h after injection compared with that in the control, vehicle groups. A subsequent decrease in feeding relative to control levels was observed in the 4-8h period after injection of CART into ARC. The groups fed the high fat diet exhibited a clear increase in food intake following injection of CART into non-fasted diabetic rats’ARC. QRT-PCR demonstrated that diabetic rats fed the high fat diet exhibited a clear increase in the CART mRNA/β-actin mRNA ratio, further enhanced that CATR may have special way to high fat diet.3.3 Effects of hypothalamic injection of NPY and CART on food intake and effects of CART on hypothalamic neuropeptide releaseIn fasted rats, injection of NPY into ARC of diabetic rats significantly increased feeding in 1-2h and 2-4h after injection compared with that in the control, vehicle groups. However, the difference between the regular chow and high fat diet were not obvious. NPY stimulated feeding was through carbohydrate intake which is most important. A ratio of carbohydrate in regular chow was 3 times higher than the high fat diet, thus, NPY may be the reflection of carbohydrate storage. Group was fed with high fat diet for 2 weeks, combination injection of CART and NPY, the food intake was much higher than the NPY group or the group was fed with regular chow for 2 weeks, combination injection of CART and NPY, further enhanced that CATR may have special way to high fat diet. Double fluorescence immunohistochemistry in the PVN of the type 2 diabetic rats showed that immunoreactivity for NPY and CART in overexpressed neurons in PVN, demonstrating colocalization of two molecules in the PVN neurons. There may be having the connection between NPY and CART. Exposure of hypothalamic explants to 0.4, 4 and 40 nmol/L CART significantly increased NPY-IR. There was a significantly increase in AGRP-IR and CRH-IR following exposure of the explants to 4 nmol/L CART. The release ofα-MSH from hypothalamic explants was not altered after exposure of the exlants to 0.4, 4 and 40 nmol/L compared with basal release. It’s possible that CART could stimulate the release of the hypothalamic orexigenic neuropeptides.3.4 Effects of PKA/CREB-mediated signalingVarious stimulators and inhibitors of the PKA pathway were administered, and CART mRNA and peptide, as well as p-CREB, levels in the diabetic rat ARC. In situ hybridization demonstrated that PKA stimulators forskolin increased CART mRNA levels, showed dose dependent manner, an effect attenuated by the inhibition of PKA （Rp-cAMPs）. Rp-cAMPs alone reduced mRNA levels compared with controls, indicating a tonic regulation by PKA on CART expression. Western blotting indicated that forskolin significantly increased CART peptide levels in a manner consistent with the observed increase in CART mRNA levels （P<0.05）. Intra-ARC forskolin administration significantly increased p-CREB levels while simultaneously decreasing the amount of CREB protein in the diabetic raGt ARC. Inhibition of PKA with Rp-cAMPs attenuated p-CREB expression.3.5 Effects of ERK signaling pathways activated by calcium influx in the hypothalamus neurons phosphorylation by CART3.5.1 Identification of hypothalamus neuronsThe hypothalamus neurons were grown very fast, there were little Glial cells. During the course of neuron growth we measured the axis length and the cell body area of the cultured neurons. 4-6 days, the cell body area and process length of nerve cells reached their highest levels. We used NF-200 to identify the cultured neurons, then calculated the percentage of the neuron that is 96.17±0.06%。3.5.2 Effects of CART on the phosphorylation of the hypothalamus neuronsWestern blotting showed that CART induced the activation of ERK 1/2 in a time- （5-90min） and dose-（1-100nmol/L） dependent manner in hypothalamus neurons. The CART effect was blocked by MEK inhibitor U0126, indicating the involvement of the upstream kinases, MEK 1/2.3.5.3 CART enhanced the [Ca²⁺]i of the hypothalamus neuronsD-hank’s and DMSO were as negative control could not affect the change in [Ca²⁺]i. In Ca²⁺-free external circumstance, exposure of the hypothalamic neurons to 100 nmol/L CART produced a rapid and significant increase of [Ca²⁺]i or unregulated fluctuation. Pretreatment of the hypothalamus neurons with MEK inhibitor U0126 30min, 100 nmol/L CART on [Ca²⁺]i was abolished, there was no statistical difference, indicated that ERK1/2 pathway processed in stimulating the hypothalamus neurons [Ca²⁺]i by CART.3.6 CART regulates islet hormone secretion and is expressed in theβcells of type 2 diabetic rats3.6.1 Effects of CART on insulin secretion from INS-1 cellThe cell density of the cultured INS-1 cell was above l×106/ml, most of the cells were showed in compressed irregular polygons. At 16.7 mmol/L glucose, 100 nmol/L CART evoked significantly augmentation of GLP-1 stimulated GSIS. The cAMP levels in cells treated with GLP-1 together with CART were higher than in cells treated with GLP-1 alone. CART potentiated cAMP-enhanced GSIS.3.6.2 Effects of CART on islet hormone secretion from isolated isletsEvery rat purification for the islet number was 303±44, DTZ staining demonstrated that the islet purity>85%, and AO-PI staining demonstrated that the islet survival rate>95%. To explore whether PKA-dependent or -independent mechanisms account for the observed effects, we used the PKA inhibitor H89. H89 completely abolished the potentiating effect of CART on insulin release.3.6.3 Effects of Sitagliptin treatment on oral glucose tolerance test, blood glucose, GLP-1 and insulin levelsA single dose of Sitagliptin （a dose-related manner） has been demonstrated to improve glucose tolerance and increase insulin secretion after an oral glucose challenge in STZ induced diabetic rats. Sitagliptin could increase active GLP-1 compared with the control and vehicle group in dose dependent manner. For the 1, 3, 10mg/kg doses, were significantly increased the plasma insulin levels compared with the control and vehicle groups.3.6.4 Effects of Sitagliptin on histopathology in type 2 diabetic rats isletHematoxylin-eosin （HE） staining was used to observe pancreatic histopathology ofβcells. Compared with the normal rats, type 2 diabetic rats’islet area significantly reduced andβcell number were obviously reduced. After Sitagliptin treatment, the islet area andβcell number were obviously increased, in a dose dependent manner.3.6.5 Expression of CART-IR、Insulin-IR、Glucagon-IRFluorescence immunohistochemistry for CART in control rats showed that CART immunoreactivity was largely restricted to theδcells and pancreatic neurons. In type 2 diabetic rats, in addition toδcells and neurons, a great portion of theβcells was CART immunoreactive. Middle and high dose of Sitagliptin treatment, the number of CART- immunoreactiveβcells were reduced compared with the vehicle. High dose of Sitagliptin treatment, CART- immunoreactivity was reduced to a level comparable with the controls. Double fluorescence immunohistochemistry for Insulin and Glugacon in control rats showed that Insulin immunoreactivity was largely restricted to theβcells and Glugacon immunoreactivity was largely restricted to theαcells. Vehicle group demonstratied that colocalization of two molecules in theβcells.3.6.6 Effects of CART on type 2 diabetic rats’βcellTEM examination revealed that labeling for CART was present in the secretory granules ofβcells andδcells. In theβcells the gold particles were mainly localized to the electro-lucent halos of the granules, between the dense core and the limiting membrane. Negative control displayed no CART labeling. In theδcells the gold particles were mainly localized to the electro-lucent dense core, not between the halos and the limiting membrane.4. Conclusions:4.1 Rats with hyperlipidemia were successfully induced with the high fat diet; type 2 diabetic rats were developed by injection low dose streptozotocin due to parts ofβcells injury. The groups fed the high fat diet exhibited a clear increase in food intake following injection of CART into ARC. These data provided evidence that hypothalamic CART has an orexigenic effect. Diabetic rats fed the high fat diet exhibited a clear increase in the CART mRNA, further enhanced that CATR may have special way to high fat diet.4.2 CART and NPY are the mediators of the regulation of food intake. CART stimulated the NPY-IR in PVN of the diabetic rats, indicated that the connection between CART and NPY. CART could stimulate the release of the hypothalamic orexigenic neuropeptides. 4.3 Inactive CREB which is associated with the CRE site of the CART promoter, to form transcriptionally active pCREB. PKA/CREB pathway may be involved in regulating CART mRNA and peptide levels.4.4 CART induced the activation of ERK 1/2 in a time- and dose- dependent manner in hypothalamus neurons. ERK1/2 pathway processed in stimulating the hypothalamus neurons [Ca²⁺]i by CART.4.5 The results of INS-1 cells and isolated islet cells demonstrated that it could be explained possibly a stronger ensuring activation of the PKA-dependent pathway. Sitagliptin （a dose-related manner） has been demonstrated to improve glucose tolerance and increase GLP-1 and insulin secretion. Fluorescence immunohistochemistry and TEM examination revealed that labeling for CART was present in the secretory granules ofβcells andδcells in type 2 diabetic rats. CART peptides not only found in the brain but also found in the gut, they are referred to as‘brain-gut’peptides.