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  • We have previously shown in the

    2021-10-18

    We have previously shown in the same time frame reported here (10 min) that AG was not degraded into DAG [29] and that the half-life of AG in human serum is ∼10 min [49]. However, we cannot rule out in this study that there is not breakdown to DAG that could contribute to the levels of 125I-ghrelin measured in brain. AG breakdown to DAG can be done by numerous circulating esterases, including butylcholinesterase and carboxypeptidase, and it is possible for various esterases to compensate when one is lacking [50]. However, what contributes to the further breakdown of DAG in Fmoc-Cys(Trt)-ol is not known. Whether hAG and mAG are hydrolyzed at different rates in our mice is unknown and may provide another mechanism for the differences we have detected in transport. To gain insight into brain regions that are involved in the central action of ghrelin, we determined the regional transport of the ghrelin peptides throughout the brain. While the overall accumulation of each ghrelin peptide varies for the brain regions, there is a significant difference in regional accumulation with greater levels present in the hypothalamus compared to the hippocampus and cortex. The differences in peptide accumulation for each brain region are likely due to recognition of the isoform with the transporter. The brain region with the highest level of each ghrelin peptide, regardless of species, was the olfactory bulb. We previously reported that the GHSR1a is present in olfactory circuits in the brain; ghrelin enhances food-seeking behavior by stimulating exploratory sniffing and increasing olfaction sensitivity [15]. Our finding is similar to the previous report by Diano et al. that the olfactory bulb (along with occipital Fmoc-Cys(Trt)-ol cortex) exhibits the highest uptake of peripherally injected radiolabeled human ghrelin in the mouse brain [51]. We are unaware whether fasting induces changes of ghrelin transport specific to the olfactory bulb. If long-term fasting is altering the structure of the BBB or c-Fos expression, ghrelin transport could also be altered. For most ghrelin forms, there was no effect of genotype on regional distribution. However, for hAG, there was a significant effect of genotype; the Ghsr null mice had consistently lower levels in each brain region compared to WT. Since only a single time point was taken to collect these data, we cannot determine if the decreased level is due to a decrease in binding to the GHSR, altered efflux, or overall transport. Based on the finding that hDAG BBB transport rate is greater than the other previously characterized ghrelin forms, we wanted to further characterize the transport characteristics of hDAG. We found hDAG was intact in mouse serum yet brain hDAG was ∼60% intact by 10 min. We also observed nearly complete transfer of hDAG across the brain endothelial cell and into the brain parenchyma. Lastly, we investigated the saturability of hDAG transport. At two different doses of unlabeled hDAG, 1 μg and 10 μg, the amount of 125I-hDAG present in the brain was not statistically different. These data suggest this system is not saturable at these doses. These results are similar to the data reported previously [29].
    Conclusions In conclusion, our results show for the first time the GHSR is not solely responsible for ghrelin transport across the BBB. At this time, it is unclear whether the two forms (AG and DAG) are transported by the same protein. In addition, we show ghrelin levels are highest in the olfactory bulb, regardless of the form of ghrelin. We also newly characterized hDAG transport characteristics in addition to comparing the results from mDAG, hAG, and mAG BBB transport to previously reported literature [29]. Both human and mouse DAGs are transported faster across the BBB compared to AG while human ghrelin peptides are transported more efficiently than mouse ghrelin. The information gained from these results aid in better understanding the gut-brain communication and provide new foundations for future investigations to connect these findings with physiological functions of ghrelin.