Effects Of Acidic Fibroblast Growth Factor Fragments On Nocturnal Feeding In Rats By Intracerebroventricular

Fibroblast growth factors (FGFs) belong to a large family of heparin-binding polypeptide growth factors, including acidic FGF (aFGF), basic FGF (bFGF). Of them, the best-studied members are aFGF and bFGF, which consist of a 140 amino acid sequence and a 146 amino acid sequence, respectively, have molecular masses of about 16,000 Da, and share 55% homology. AFGF promoted the proliferation of fibroblast cell. AFGF had protective effects on neuron and important organs after the emergency injury, and aFGF also had a suppressant effect on feeding behavior. But bioactive site of aFGF is still unknown. In our study, seven aFGF fragments were infused into the third ventricle of rats to investigate the active region of aFGF. These findings may be beneficial to the development of clinical applications.Aim:1. Seven aFGF fragments were infused into the III ventricle of rats to investigate the active region of aFGF that is responsible for food intake by calculating nocturnal food consumption.2. The effectiveness of peripheral administration of the active fragments were also examined by calculating nocturnal food consumption.Methods:1. Maintenance and groups: Male Wistar rats (n=162) were maintained in individual cages with artificial light illumination from 07:00-19:00 h and with room temperature of 22±2°C. The rats were randomly divided into two groups, normal control groups and aFGF fragments groups. For intracerebroventricular injection, all aFGF fragments were divided into 200ng/rat and 400ng/rat. For hypodermic injection, all aFGF fragments were divided into 80μg/kg,100μg/kg and 300μg/kg.2. Implantation of Brain Cannulas: Rats were anesthetized by pentobarbital (40 mg/kg, IP) and fixed in a stereotaxic apparatus. For ICV infusion, a 23-ga stainless steel guide cannula (18 mm long, 0.64 mm o.d., 0.39 mm i.d.) was chronically implanted into the third ventricle at stereotaxic coordinates: AP, -0.2mm; L, 0.0 with respect to bregma; and H, 7.5-8.0mm from the brain surface. A sterile stainless steel obturator (0.33 mm o.d.) with a cap was used to ensure the patency of the tip of the guide cannula.3. Infusion and calculating food intake: For intracerebroventricular infusion, a guide cannula made of stainless steel tubing was fixed into the cerebral 3rd ventricle one week before the experiments. Under no anesthesia, seven synthesized aFGF fragments aFGF-(1-15), [D-Trp6]-aFGF-(1-15), [desaminoPhe¹.D-Trp⁶]-aFGF-(1-15), [desaminoPhe¹.Lys(ε-myristyl)16]-aFGF-(1-16), [Lys(ε-myristyl)¹⁶]-aFGF-(1-16), [D-Trp⁶.Lys(ε-myristyl)16]-aFGF-(1-16) and [Ala16]aFGF-(1-29) were injected into the 3rd ventricle of rats in the period 18:30-19:00. Food in the animal food boxes were weighed at 19:00, 22:00, 7:00 for calculating food consumption by rats in 3 hours and 12 hours. Then the two active aFGF fragments aFGF-(1-15) and [Ala¹⁶]aFGF-(1-29) were injected into the subcutaneous tissue of rats in the period 18:30-19:00, and also for calculating nocturnal food consumption.4. Histology: After the experiments, the rats were deeply anesthetized with pentobarbital, and pontamine sky blue (2%) was infused into the third ventricle (10 u1). The rats were then transcardially perfused with physiological saline followed by 10% buffered formalin, and the brains were removed, cut coronally into 50-um thick sections, and stained by eresyl violet. The marking site and size were determined in each animal under a light microscope.Results:1. aFGF-(1-15) (200ng/rat) had no effect on the feeding, that of aFGF-(1-15) (400ng/rat) suppressed the food intake(3h: 3.0±0.2 vs 2.1±0.2; 12h: 18.5±0.5 vs 16.1±0.5, P <0.01).2. [Ala16]aFGF-(1-29) suppressed the food intake not only dose of 200ng/rat(3h: 4.9±0.2 vs 3.4±0.2; 12h: 19.3±1.2 vs 17.3±1.1, P<0.01) but also dose of 400ng/rat (3h: 3.6±0.1vs 1.6±0.2; 12h: 19.9±0.8 vs 16.4±1.6, P <0.01).3. Other five aFGF fragments had no effect on the feeding in the dose of 200ng/rat and 400ng/rat (P >0.05).4. For hypodermic injection, [Ala16]aFGF-(1-29) (300μg/kg) suppressed the food intake (3h: 3.9±0.2 vs 2.1±0.3; 12h: 19.8±0.5 vs 11.2±0.8, P <0.01) while others had no effect (P >0.05).Conclusions:1. These findings suggest the amino-terminal portion of aFGF is active in food intake suppression.The replacement of cysteine residue by alanine is important in some amino-terminal aFGF fragments.2. Other aFGF fragments, in which glycine at position 6 was replaced with D-tryptophane, phenylalanine at position 1 with desaminoPhe, cysteine at position 16 with Lys(ε-myristyl) had no effect on nocturnal feeding in rats.3. Peripheral administration of one fragment was also effective on nocturnal feeding in rats. Leptin is a white adipocyte-derived hormone that plays a key role in the regulation of food intake, body weight, energy expenditure and neuroendocrine function. Leptin involved in energy balance, food intake, and the functional activities of the digestive tract. These biological effects of leptin are exerted through the leptin receptor (Ob-R). Feeding behavior is implemented by the stomach and food intake is controlled by gastric motility. Therefore how the Leptin regulates gastrointestinal motility and control the food consumption must be well studied.It has been known that dorsal vagal complex (DVC) is a very important part in the regulation of gastrointestinal motility in the central nerve system, and hypothalamus is the superior central of the dorsal vagal complex. Researches have shown that Leptin regulates feeding behavior mediated by the regulation of proopiomelanocortin (POMC) expression in the hypothalamus. Then, little is known about potential leptin signaling to regulate gastrointestinal motility in POMC neurons located in the DVC. Therefore it will be investigated in our study.Aim:In our study, leptin was infused into the lateral cerebral ventricle of rats to investigate gastrointestinal motility and to observe the expression of POMC and OB-R in the dorsal vagal complex. Methods:1. Maintenance and groups Female SD rats weighing 180–220g (n=73) were maintained in individual cages with artificial light illumination from 07:00-19:00 h and with room temperature of 22±2°C. The rats were randomly divided into two groups, normal control groups and infused leptin groups. Leptin groups were divided into three subset group (1, 3, 5h).2. Implantation of Brain Cannulas and InfusionRats were anesthetized by 10% Chloral Hydrate (0.3ml/100g, IP) and fixed in a stereotaxic apparatus. For the lateral cerebral ventricle infusion, a 23-ga stainless steel guide cannula (13mm long, 0.64mm o.d., 0.39mm i.d.) was chronically implanted into the lateral cerebral ventricle at stereotaxic coordinates: AP, -0.8mm; L, 1.5 with respect to bregma; and H, 3.5mm from the brain surface. A sterile stainless steel obturator (0.33mm o.d.) with a cap was used to ensure the patency of the tip of the guide cannula. For intracerebroventricular infusion, a guide cannula made of stainless steel tubing was fixed into the cerebral 3rd ventricle one week before the experiments.Under no anesthesia, the rats were fasted for 20-24 hours, and then leptin (3.5μg/μl) was injected into the lateral cerebral ventricl through the implanted guide cannula. Same volume saline were injected as the control.3. Internal Fixation of the Ventricles and ImmunohistochemistryThe rats were anesthetized 1, 3, 5 hours after injection, a physiological saline followed by 4% buffered formalin were then perfused through left artrium, and brain stem including DVC were removed, cut coronally into 5-μm thick sections. The location of DVC were observed by hematoxylin and eosin stain. The expression of OB-R and POMC in DVC (n=5 in each group) were observed by immunohistochemical method. 4. The Measurement of Gastrointestinal MotilityFifteen minutes before sacrificed, the rats (n=28, n=7 in each group) received 2ml phenol red intragastrically at the dose of 500 mg/L, the stomach were removed and washed with with water 20ml, then added 0.5 mol/L NaOH ,20 ml,the solution were then measured absorbance value of 560nm on spectrophotometer to determine the rate of gastric emptying. Also fifteen minutes before sacrificed the rats (n=24, n=6 in each group) received 5% ink intragastrically to measure the rate of intestinal transit.Results1. The rate of gastric emptying in leptin groups decreased significantly compared with normal control groups [1h, 3h, 5h: (54.7±8.3)%. (54.6±9.3)%. (57.4±8.9)% vs (70.0±6.1)%, P <0.05]. But the rate of intestinal transit had no differences between leptin groups and normal control groups [1h, 3h, 5h: (41.1±4.9)%. (49.5±13.6%. (43.6±5.5)% vs (43.0±6.3) %, P >0.05].2. By immunohistochemical method, the expression of OB-R protein in DVC increased in leptin groups (P <0.05) while the expression of POMC had no change compared with normal control groups (P>0.05).Conclusions1. Our study suggest that leptin injected intracerebroventricularly inhibits the rate of gastric emptying in rats and had no effect on the rate of intestinal transit.2. Unlike in ARC, this procedure does not because of the simulation of POMC neurons in the DVC, but may through the leptin receptor (Ob-R) directly in DVC.