Central neuronal pathways influenced by Adipokines and insulin
Creators: name: Alsuhaymi, Naif
Publisher: name: RMIT University
Year: 2024
Subjects: classificationCode: 320101
subject: Cardiology (incl. cardiovascular diseases)
classificationCode: 320903
subject: Central nervous system
classificationCode: 340102
subject: Bioassays

description: Resistin, leptin, and also insulin have roles in cardiovascular function, feeding and energy balance; and can act in the brain nuclei known to be important for mediating these functions. Central sites of action for resistin are not well understood, in contrast to leptin or insulin. Moreover, in high fat diet (HFD), resistin, leptin, and insulin are increased, but little is known regarding the interactions between these hormones in the brain. Resistin and leptin are both involved in inflammatory responses in the periphery and it is known that leptin is involved in inflammatory actions in the brain, so we speculated that resistin may have similar effects in the brain. Therefore, this project investigated the distribution of neurons and their neurochemical content in the brain that are activated by centrally administered resistin, leptin or insulin alone; or a combination of these hormones, in rats fed a HFD compared to a normal diet (ND). In addition, this project explored the effects of leptin and resistin in brain inflammation. Methods Male Sprague-Dawley rats were fed a high fat diet (22% fat) or normal diet. Anaesthesia was induced with 2-5% isoflurane in O2 to allow femoral artery and vein cannulation. Urethane (1.4-1.6 g/kg initially, followed by additional doses of 0.05 ml, as required) was used to maintain the anaesthesia. The rats received intracerebroventricular (ICV) saline (5 μl), leptin (7 μg), resistin (7 μg), or insulin (500 mU), or a combination of resistin and leptin, leptin and insulin, or resistin and insulin. Three hours later, the brains were perfused and removed. Immunohistochemistry was performed on these brains to detect Fos protein, a marker of activated neurons; or CD11b (clone OX42), a marker for microglia. Dual-label immunohistochemistry was performed to detect activated neurons containing tyrosine hydroxylase (TH), a marker for catecholamines; or tryptophan hydroxylase, a marker for serotonin; or orexin. In separate studies, the brains were immediately removed and divided into blocks of the hypothalamus, midbrain, and ventral and dorsal medulla to investigate the gene expression of cytokines (TNF-α, IL-1β, and IL-6) using real-time polymerase chain reaction (RT-PCR). Results In chapter three, the results show that in the arcuate nucleus (ARC), paraventricular nucleus (PVN) and lateral hypothalamic area (LHA), leptin or resistin administered alone or in combination significantly increased the number of Fos-positive neurons; and this occurred in the lamina terminalis only when leptin and resistin were combined. Only in the ARC was the response to resistin and leptin combined significantly greater than for each hormone alone. In the periaqueductal gray (PAG), resistin alone also significantly increased the number of Fos-positive neurons compared to control and this was greater than the combination of resistin and leptin. Interestingly, in the raphe pallidus nucleus (RPA), when both leptin and resistin were administered, the number of Fos-positive neurons was significantly reduced compared to either hormone alone. In chapter four, the results show that following resistin or leptin alone or in combination in rats fed the HFD, there were no significant increases in the number of Fos-positive cell nuclei in the PVN, ARC, and the LHA. This contrasted with observations in rats fed a ND. In the organum vasculosum of the lamina terminalis (OVLT) and median preoptic nucleus (MnPO) of HFD rats, there were significantly smaller numbers of Fos-positive cell nuclei compared to ND following the combined administration of resistin and leptin. In the PAG, rostral ventromedial medulla (RVMM) and nucleus tractus solitarius (NTS) of HFD rats, there were significantly less Fos-positive cell nuclei compared to ND following resistin alone. In chapter five, resistin and leptin alone or in combination increased activation of catecholaminergic neurons in the PVN in ND and in the NTS and rostral ventrolateral medulla (RVLM) in both ND and HFD diets, and increased activation of orexinergic neurons in the LHA in ND. Only resistin and leptin in combination increased activation of serotonergic in the RVMM, but only leptin alone increased activation of serotonergic neurons in the dorsal raphe nucleus (DR), in rats fed a ND. In chapter six, compared to control, insulin alone or combined with leptin increased neuronal activation only in the PVN of rats fed a ND, and a HFD attenuated this effect of insulin alone. Neuronal activation in the PAG was reduced in ND and HFD. A reduction was also observed in the NTS and RVLM in HFD but not in ND. Insulin alone or in combination with leptin increased activation of catecholaminergic neurons in the RVLM in both diets and in the NTS in ND. Insulin combined with leptin increased activation of catecholaminergic neurons in the NTS in HFD. In chapter seven, compared to control the combination of insulin and resistin resulted in significantly less neuronal activation in the PAG in both diets, and in the NTS, RVLM, and RVMM in HFD. It was also found that compared to resistin alone, the combination resulted in less neuronal activation in the PVN, PAG, NTS, and LHA of rats fed a ND; and in the NTS and RVLM of HFD rats. Only in the PVN of ND rats was there less neuron activation compared to insulin alone. There was little effect on catecholaminergic, serotonergic, and orexinergic neurons observed. In chapter eight, resistin and leptin alone or in combination significantly increased microglial activation only in the PVN of rats fed a ND. No other brain regions examined showed significant activation of microglia. Gene expression of tumor necrosis factor alpha (TNF-α) in the midbrain and ventral medulla, and interleukin 1 beta (IL-1β) in the midbrain and dorsal medulla of rats fed a HFD were increased with resistin. The combination of resistin and leptin increased TNF-α gene expression in all brain regions that were examined except the hypothalamus, in addition to increased IL-1β and interleukin 6 (IL-6) gene expression in the ventral medulla of rats fed a ND. In conclusion, this thesis provides novel information and knowledge regarding the potential sites of action of resistin and the nature of the neurons activated in the brain. The potential sites of action in hypothalamic nuclei include the ARC, PVN, and LHA, and these are similar to that of leptin. In such regions, resistin was found to mainly activate catecholaminergic and orexinergic neurons, as observed with leptin. Furthermore, these responses can be reduced by a HFD. Additionally, there is evidence that resistin contributes to brain inflammation.