["Acta Physiologica, Volume 231, Issue 3, March 2021. ", "\nAbstract\n\nAim\nMetabolic sources switch from carbohydrates in utero, to fatty acids after birth and then a mix once adults. O‐GlcNAcylation (O‐GlcNAc) is a post‐translational modification considered as a nutrient sensor. The purpose of this work was to assess changes in protein O‐GlcNAc levels, regulatory enzymes and metabolites during the first periods of life and decipher the impact of O‐GlcNAcylation on cardiac proteins.\n\n\nMethods\nHeart, brain and liver were harvested from rats before and after birth (D‐1 and D0), in suckling animals (D12), after weaning with a standard (D28) or a low‐carbohydrate diet (D28F), and adults (D84). O‐GlcNAc levels and regulatory enzymes were evaluated by western blots. Mass spectrometry (MS) approaches were performed to quantify levels of metabolites regulating O‐GlcNAc and identify putative cardiac O‐GlcNAcylated proteins.\n\n\nResults\nProtein O‐GlcNAc levels decrease drastically and progressively from D‐1 to D84 (13‐fold, P < .05) in the heart, whereas the changes were opposite in liver and brain. O‐GlcNAc levels were unaffected by weaning diet in any tissues. Changes in expression of enzymes and levels of metabolites regulating O‐GlcNAc were tissue‐dependent. MS analyses identified changes in putative cardiac O‐GlcNAcylated proteins, namely those involved in the stress response and energy metabolism, such as ACAT1, which is only O‐GlcNAcylated at D0.\n\n\nConclusion\nOur results demonstrate that protein O‐GlcNAc levels are not linked to dietary intake and regulated in a time and tissue‐specific manner during postnatal development. We have identified by untargeted MS putative proteins with a particular O‐GlcNAc signature across the development process suggesting specific role of these proteins.\n\n"]