| IntroductionGastric Iinhibitory Peptide (GIP), also known as Glucose-dependent Insulinotropic Polypeptide, is a 42 amino acid-long peptide hormone synthesized in and released from duodenal and jejunal K-cells in response to the ingestion of nutrients, especially dietary lipids in which it facilitates the disposal of both glucose and fat. Of the incretins, GIP has received much interest in the role of regulation of glucose and lipid metabolism.By binding to a seven-transmembrane domain G-protein-coupled receptor expressed in adipose, GIP achieves effects such as regulation of adipocyte differentiation, increase in adipocyte glucose uptake, modulation of adipocyte lipolysis and reesterification and promotion of the storage of triglycerides by increasing the activity of lipoprotein lipase. Moreover, it has been shown that GIP is a key molecule linking overnutrition to obesity. Previous reports about GIP receptor knockout mice resistant to obesity suggest that inhibition of GIP signaling might be a novel target for anti-obesity drug development. The administration of the GIP-specific receptor antagonist and targeted ablation of GIP-producing cells in diet-induced obese mice have also been confirmed to improve metabolic activity. Collectively, inhibition of GIP action may offer exciting possibilities for the development of future therapies available for patients with obesity-related problems.Conventionally, vaccines are usually used to induce specific immune responses against foreign, pathogen-derived structures. In marked contrast, vaccination against self-molecules is an unusual non-traditional method. Using this novel approach of vaccinating immune bodies against their own endogenous GIP may prove a promising design for combating obesity and related comorbidities. In this respect, the data presented by Fulurija A, Irwin N and colleagues support the neutralization of biologically active GIP by immunopharmacotherapy. The potential advantages of vaccines in the treatment of non-communicable and chronic diseases were thought be the beneficial long-term effects of a relatively short-term treatment, which required less compliance at the same time. However, the treatment may become more complicated with a GIP-targeted vaccine. It has been shown that GIP receptors are widely distributed in the brain and that they may modulate neural progenitor cell proliferation and protect neurons from toxic effects. To a certain extent, GIP may regulate brain function and behavior. Recently, it has been demonstrated that the high circulating GIP concentration brings about significant changes in behaviors. One example is that GIP-overexpressing transgenic mice have better sensorimotor coordination and memory recognition. Therefore the question remains that if immunization methods are used to change the circulating physiological GIP concentration, will there be a resultant change in brain function and behavior.Here, we constructed GIP vaccine and first observed that the influence of active vaccination of mature rats with GIP immunoconjugates on body weight as well as on brain function and behavior.ObjectiveTo construct GIP vaccine. Virus-like particles (VLPs) formation by modified Hepatitis B virus core (HBc) or keyhole limpet hemocyanin (KLH), acting as a kind of immune-enhancing vaccine carrier, was fused with the specified epitope of gastric inhibitory peptide(GIP) to construct GIP vaccine (HBc-GIP and GIP-KLH) and the immune effects were investigated. To investigate the effects of active immunisation against GIP on weight gain, behavior and brain function after active immunization against GIP in high-fat-diet SD rats, which may provide theoretical and experimental basis of immune therapy of obesity.MethodsGIP (1-12a.a.) cDNA was synthesized and fused to HBc VLPs (1-144a.a.) cDNA. Through the bacterial expression and purification processes, GIP vaccine attached to the VLPs carrier derived from HBc was constructed. Hapten GIP peptide (GPRYAEGTFISDC), added a cysteine in the N-terminal region of GIP-(1-12) amides segment, were synthesized by using solid phase peptide synthesis. And Synthetic hapten peptide was purified by HPLC, and coupled to the carrier keyhole limpet hemocyanin (KLH), its reaction with m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), and yielding immunoconjugates GIP-KLH. The immunogenicity of HBc-GIP and GIP-KLH vaccine in rats was studied.By monitoring of body weight, open field test (OFT), Morris water maze (MWM) test, 18F-fluorodeoxyglucose (FDG)-Positron emission tomography/computed tomography (PET/CT) examination, the terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) or proliferating cell nuclear antigen (PCNA) method, the differences in weight gain, behavior and brain function between the GIP-KLH-treated group and the KLH-treated group were investigated.Results:1. GIP vaccine attached to the VLPs carrier derived from HBc was successfully achieved. Vaccination with DNA Encoding HBc-GIP fusion protein (pVAX1-HBc-GIP) was also gotten. Both the immunization with DNA vaccine (HBc-GIP) or DNA vaccine (pVAXl-HBc-GIP) alone and the combination of DNA and adjuvanted protein vaccination resulted in high, peptide-specific IgG antibody. HBc-GIP and pVAX1-HBc-GIP vaccine displayed a significant strong immunogenicity, whereas control animals failed to exhibit this change. Immunization strategy of prime-boost immunization approach using a combination of DNA and protein vaccine (pV-HBc-GIP*2+HBc-GIP*2) resulted more higher antibody than using DNA or ptotein vaccine alone.After the third immunization, immunized animals induced higher titers of anti-GIP antibodies. Following the vaccination protocol, experimental rats had further increases in antibody levels. Increased immune responses in high-fat diet-induced SD rats by DNA vaccination combined with protein vaccine showed good results.2. GIP vaccine attached to the KLH carrier was successfully achieved. On day 7 after the second immunization, GIP-KLH-immunized animals induced higher titers of anti-GIP antibodies compared to control rats (KLH-immunized animals). Following the vaccination protocol, experimental rats had further increases in antibody levels, whereas control animals failed to exhibit this change. Increased immune responses in high-fat diet-induced SD rats by GIP-KLH vaccination showed good results.3. Although no significant differences were found in food intake (p>0.05), differences in increased body weight emerged among groups over the study period. GIP-KLH-vaccinated rats displayed significantly reduced body weight gain compared to KLH-treated rats. Even though there was no difference in the initial body weight, GIP-KLH-vaccinated rats began to gain weight more slowly after a 63-day high-fat feeding compared to other vaccinated rats. At the end of the experiment (98 days after the first vaccination), these experimental rats had gained 48g less weight than control animals (17% reduction in weight gain from the beginning). Although GIP is a key incretin in metabolism, fasting plasma glucose levels and serum insulin levels were unaffected after vaccination in our study (p>0.05).4. In OFT, Compared with control rats, movement distance (P<0.01), movement speed (P <0.01), active times (P<0.05), the number of activities (P<0.01) the time in the center squares(P< 0.05) decreased and rest time increased (P<0.05) significantly in GIP immunised rats. Thus, the GIP-KLH-immunized rats'automated locomotor ability and the activities of the center squares were significantly decreased compared to the KLH-treated group. Therefore, it is likely that GIP vaccine could lead to behavioral side effects. In Morris water maze (MWM), all rats learned to locate the hidden platform as evidenced by a significant decrease in escape latency and swim distance (F=6.5; P=0.001 and F=5.3; P=0.002, respectively) and a significant increase in swim speed (F=3.3; P=0.024) over the 4-day trial period. However, no statistical difference was shown between GIP-KLH-immunized rats and KLH-treated rats with regard to the previously mentioned data (P>0.05). In the retention test (probe trial) 4-days later, neither GIP-KLH-immunized nor KLH-treated rats exhibited a better retention over the other or had more crossings over the previous location of the platform (P>0.05).5. In the vivo, the 98th day after application of vaccine, brain glucose utilization assessed by 18F-FDG PET was reduced in hippocampus and cerebral cortices in the GIP-KLH-treated group compared to the KLH-treated group (P<0.05). However, there was no significant difference in olfactory bulbs, or cerebella (P>0.05).6. Granule cells were marked by the TUNEL or PCNA method in the areas of hippocampal dentate gyrus of GIP-KLH-immunized rats and KLH-treated rats. TUNEL-positive (brown nuclear staining) apoptotic cells and PCNA-positive cells (brown nuclear staining) were found in both groups. But, Comparing the number and intensity of positive cells between two groups, we noticed a significantly larger number of marked cells by TUNEL or PCNA method in the hippocampus of the animals treated with GIP-KLH vaccine.Conclusion:HBc(1-144a.a.) or KLH acts as a kind of immune-enhancing vaccine carrier for GIP successfully. Both HBc-GIP and GIP-KLH vaccine are able to overcome GIP-specific B cell unresponsiveness. Active immunisation against GIP results in high, GIP-specific antibody titers. The results encourage further work towards the development of GIP vaccine against obesity. At the same time, active vaccination of mature rats with GIP immunoconjugates may be associated with significant changes in not only body weight, but also brain function and behavior.
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