Short-Term High-Fat Diet Promotes Increased Lysine Crotonylation in Cerebral Cortex

Protein lysine crotonylation is a newly discovered protein post-translational modication (PTM), which has been associated with cellular metabolism, cell cycle, gene transcription, DNA damage response. However, its potential roles related to human central nervous system diseases remain largely unknown. In the present study, we observed a signicant elevated lysine crotonylation in a screening of nine lysine acylations in cortex tissues of HFD-fed mice after short-term overfeeding. On the base of previous reports and molecular weight of proteins, we also speculate that actin, ERK2 or GAPDH and CDK1 might be modied by lysine crotonylation (KCr). Taken together, our ndings highlight a potential role of protein lysine crotonylation in HFD-induced brain disorders and as possible therapeutic candidates in the future.


Introduction
In living organisms and individual cells, post-translational modi cations (PTMs) of proteins are crucial for regulation diverse cellular functions, such as DNA replication, transcription, tissue differentiation, apoptosis, in ammation and so on (Berdasco and Esteller, 2010;Lee, 2013). To date, more than 500 discrete types of PTMs across all 20 protein amino acids have been identi ed (Keenan et al., 2021). In addition to extensive studies of common PTMs, like phosphorylation and acetylation (Acetyllysine, KAc) (Verdin and Ott, 2015), several novel protein acetylation, such as, Lysine malonylation (Malonyllysine, KCr modi cation might become a potential intervention target to ght against HFD-related brain diseases.

Reagents
Congo red (C6767) and Coomassie Blue Fast Staining Solution (P0017) were purchased from Sigma-Aldrich and Beyotime Biotechnology respectively. All antibodies used in this study are listed in the Key Resources Table 1.
Animals, High-fat diet C57BL/6J wild type male mice (8-weeks old) were provided by Gem Phar ma tech Co., Ltd (Nanjing, China). Low fat and no sugar Chow diet (TP23100) and High fat diet (HFD, TP23103, ~45% of energy) were provided by Trophic Animal Feed High-tech Co., Ltd (Nantong, China). After 7 days adaptation, mice were randomly divided into three groups (n=3 mice/group). Chow diet groups were served as controls.
Other two groups were fed with HFD for 7 days and 21days respectively. Mice were subsequently humanely sacri ced under sodium pentobarbital anesthesia. Cerebral cortex was collected and homogenized in RIPA lysis buffer (P0013B). The homogenized tissues were centrifuged at 13,000 rpm for 30min and the supernatants were kept for later steps. Mouse studies were reviewed and approved by the

Statistical analysis
All data were reported as Mean ± SD (standard deviation). All statistical analyses were performed using GraphPad software (GraphPad Prism version 8.00, San Diego, CA). Differences between two groups were tested by unpaired two-tailed Student's t-test. The signi cance level were set when *P < 0.05.

Results
Short-term HFD feeding increased crotonyllysine levels in mouse cerebral cortex To investigate the changes of protein lysine acylations in brain after 21 days short-term HFD, nine types of lysine acylations were detected by western blotting in mouse cerebral cortex lysates. We routinely loaded 15µg total protein onto each line in chow diet and HFD groups, then equal loading was veri ed by Congo red Nitrocellulose staining and Coomassie blue gels staining (Fig. 1A, B). Subsequently, we observed no changes of KMal, KSu, KLa, KPr, KBu, KAc, KHb and KGl in HFD groups compared to chow diet (Fig. 1C-J). Furthermore, although KLa in one group of HFD decreased remarkably, but no signi cant changes were observed in cerebral cortex of HFD compared to chow diet mice (Fig. 1F). Together, our results suggest that 21 days HFD does not regulate the aforementioned eight modi cations of lysine acylations in mouse cerebral cortex. However, we found occasionally that signi cant elevation of KCr was observed in HFD groups after 21 days overfeeding compared to chow diet (P < 0.01, Fig. 1K-L). In addition, to explore whether the increase of KCr levels also could occur at day 7 after HFD, we detected protein KCr and observed signi cant elevation of KCr expressions in HFD groups compared to chow diet (P < 0.05, Fig. 1M-N). In conclusion, our research indicate that KCr elevation might play major roles in multiple biological processes in mouse cerebral cortex exposed to short-term HFD.

Discussion
In the current study, we analyzed the nine types of acylation patterns in chow diet and HFD mice after short-term overfeeding. We observed an elevation of protein KCr levels in cerebral cortex tissues of chow diet and HFD animal models. Yet other eight acylation categories, such as, KMal, KSu, KLa, KPr, KBu, KAc, KHb and KGl did not demonstrate signi cantly changes in HFD groups compare with chow diet. Finally, we hypothesize that brain cortex-speci c elevation of KCr might be common in short-term HFD. the same time, we also observed two increased KCr modi cations bands adjacent to 40 and 35kDa at day 7 and 21 after HFD. Because of no available KCr antibody for immunoprecipitation, we cannot con rm the exact proteins modi ed by KCr in HFD groups. However, we speculated that KCr proteins might be actin, ERK2 or GAPDH and CDK1, according to molecular weight of these proteins and a report from high-resolution liquid chromatography-tandem MS (LC-MS/MS) (Xu et al., 2017).
In summary, elevated KCr modi cations in cerebral cortex may exert direct in uence over regulating metabolism and other unknown functions in short-term HFD. Future work in this eld should focus on the identi cation of speci c proteins related to KCr modi cations, and evaluate their roles whether these are protective or pathogenic responses in HFD-induced brain diseases.  Tables   Due to technical limitations, table 1 is only available as a download in the Supplemental Files section. Figure 1 Nine lysine acylation changes in cerebral cortex under short-term chow diet and HFD feeding.

Figures
Supernatants were collected from mice brain cortex fed by a chow diet (n=3 mice) and HFD for 7 days (n=3 mice) or 21 days (n=3 mice), which were followed by staining and blots.