br Materials and methods br Results br Discussion

Materials and methods
Results
Discussion Several peripheral hormones that are involved in the central regulation of energy homeostasis, such as leptin, insulin, and amylin [34], [35], [36], [37], [38], cross the BBB by saturable and unsaturable mechanisms. Whether these transport processes are predominantly by signaling receptor-mediated transport is debated. A novel aspect of this study was to determine whether the GHSR mediated transfer of ghrelin from blood to brain. To test this, we compared the transport rate of the human and mouse ghrelin forms in WT mice and Ghsr null mice. We found the transport rate across the BBB did not differ with the loss of GHSR. This suggests the signaling-related receptor for ghrelin is not the ghrelin transporter. This is not the first time it has been shown a peptide is transported via a different protein than the canonical signaling receptor. For example, the efflux transporter for PACAP27, a pluripotent neuropeptide, is β-F1 ATPase, a component of peptide transport system-6, and differs from the canonical signaling-related receptors, PAC1, VPAC1, and VPAC2 [39]. In addition, epidermal growth factor transporters differ from their signaling-related receptor [40]. There is consensus in the field that ghrelin is not synthesized in the A-366 [41], [42]. Therefore, the orexigenic actions of ghrelin depend on the ability of peripheral ghrelin to assess the neuronal circuits of food intake regulation. Ghrelin can gain access to the brain through the circumventricular organs (CVOs), blood-cerebrospinal fluid barrier, and the BBB [43]. We focused our research on ghrelin transport through the BBB and found that, under ad lib fed conditions, radioactive mAG has low accessibility to the mouse brain and the levels in the hypothalamus were similar to the cortex, thalamus, and whole brain. These findings raise questions about the physiologic importance of the peripheral AG transported across the BBB to reach its central targets. However, there is evidence for fasting to enhance peripheral metabolic signaling to the arcuate nucleus of the hypothalamus (ARH) [44], and we have shown enhanced BBB transport of human AG into whole brain [21]. Our previous studies also showed a numerical increase in hAG entry into whole brain in 48-hr starved mice, compared to fed mice; however, this was not statistically significant [21]. We did not examine ghrelin-mediated signaling in neurons in this study, but it is curious whether fasting would increase the BBB transport of ghrelin into the ARH and whether AG and DAG would be affected similarly. While our studies involved large, regional dissections, other studies have investigated more detailed entry into sub-regions [45], especially in the hypothalamus, and found differences in transport within this brain region. AG was able to rapidly access neurons of the ARH near fenestrated capillaries in the median eminence [45]. However, the precise transport mechanism for ghrelin into these regions has yet to be determined. Various forms of ghrelin (hAG, mAG, and mDAG) have been previously investigated in terms of BBB transport and characteristics [29]. We found the same pattern of transport rate with mDAG > hAG > mAG in the current study. Additionally, we also investigated the transport rate of hDAG in comparison to the other ghrelin forms as there are multiple CNS biological functions attributed to DAG despite its inability to activate the GHSR. Surprisingly, we found hDAG was transported approximately three times faster than the other three forms. Therefore, the new hierarchy of transport is hDAG > mDAG > hAG > mAG in mice. It should be noted, our study included males and females combined in determining the transport rate for each ghrelin peptide. Therefore, further studies should investigate a potential sexual dimorphism in the transport of ghrelin across the BBB. Human and mouse AG only differ in sequence at amino acids 11–12 [46], and human and mouse GHSR are 95% identical. To our knowledge, the binding affinity of human ghrelins to mouse GHSR and vice versa has not been addressed. The slower transport of AG could be due to the presence of its octanoyl group since the octanoyl tail allows it to circulate bound to HDL, LDL, and triglyceride-rich lipoproteins, while DAG only binds to HDL [47], [48]. It is also possible the brain-to-blood efflux system for 125I-AG contributes to the slower overall transport rate [29].