Hypothalamic astrocytes are particularly affected by energy-dense food consumption. How the anatomical location of these glial cells and their spatial molecular distribution in the arcuate nucleus of the hypothalamus (ARC) determine the cellular response to a high caloric diet remains unclear. In this study, we investigated their distinctive molecular responses following exposure to a high-fat high-sugar (HFHS) diet, specifically in the ARC. Using RNA sequencing and proteomics, we showed that astrocytes have a distinct transcriptomic and proteomic profile dependent on their anatomical location, with a major proteomic reprogramming in hypothalamic astrocytes. By ARC single-cell sequencing, we observed that a HFHS diet dictates timeand cell-specific transcriptomic responses, revealing that astrocytes have the most distinct regulatory pattern compared to other cell types. Lastly, we topographically and molecularly characterized astrocytes expressing glial fibrillary acidic protein and/or aldehyde dehydrogenase 1 family member L1 in the ARC, of which the Luiza Maria Lutomska and Viktorian Miok contributed equally to this work.

Hypothalamic astrocytes are particularly affected by energy-dense food consumption. How the anatomical location of these glial cells and their spatial molecular distribution in the arcuate nu-cleus of the hypothalamus (ARC) determine the cellular response to a high caloric diet remains unclear. In this study, we investigated their distinctive molecular responses following the expo-sure to a high-fat high-sugar (HFHS) diet, specifically in the ARC. Using RNA sequencing and proteomics, we showed that astrocytes have a distinct transcriptomic and proteomic profile de-pendent on their anatomical location, with a major proteomic reprogramming in hypothalamic astrocytes. By ARC single-cell sequencing, we observed that a HFHS diet dictates time- and cell- specific transcriptomic responses, revealing that astrocytes have the most distinct regulatory pat-tern compared to other cell types. Lastly, we topographically and molecularly characterized as-trocytes expressing glial fibrillary acidic protein and/or aldehyde dehydrogenase 1 family mem-ber L1 in the ARC, of which the abundance was significantly increased, as well as the alteration in their spatial and molecular profiles, with a HFHS diet. Together, our results provide a detailed multi-omics view on the spatial and temporal changes of astrocytes particularly in the ARC dur-ing different time points of adaptation to a high caloric diet.