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Food perception promotes phosphorylation of MFFS131 and mitochondrial…

- - - - Known metabolic adaptations in the liver rely on hepatocyte intrinsic signaling mechanisms regulated by hormones that convey the organismal energy state to the liver
        
        Claude Bernard described the dependence of hepatic glycogenolysis on signals originating from the central nervous system (CNS)
        
        Key neuronal populations (which are critical regulators of feeding behavior and adapt hepatic glucose and lipid metabolism)
        
        the arcuate nucleus of the hypothalamus (ARC) include agouti-related peptide (AgRP)- and pro-opiomelanocortin (POMC)–expressing neurons
        
        POMC neurons are now understood to be activated rapidly upon sensory perception of food as a predictive behavior
        
        sensory food perception–driven activation of POMC neurons regulates liver metabolism during innate and learned physiological responses to food perception (cephalic phase) by promoting mammalian target of rapamycin (mTOR) signaling, X-box binding protein 1 (Xbp1) splicing, and endoplasmic reticulum (ER) adaptations
        
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  - - - Rapid changes in mitochondria in POMC neurons contribute to systemic metabolic control , and melanocortin neurons in turn regulate peripheral tissue function in part through regulation of mitochondrial function
        
        Modulation of mitochondrial dynamics has substantial effects on systemic and hepatic glucose metabolism
- - - - performed two unbiased phosphoproteomic screens in liver
        
        First, we evaluated whether sensory food perception or refeeding was sufficient to alter protein phosphorylation in liver mitochondria within 30 min.
        
        To identify phosphorylation sites that are commonly regulated between food perception and refeeding, significantly regulated sites
        
        Moreover, after food perception and refeeding, we detected an increased phosphorylation of downstream targets of mTOR signaling, such as ribosomal protein S6 AND eukaryotic translation initiation factor 4B
        
        examined phosphorylation events in the mouse liver upon optogenetic POMC neuron activation.
        
        liver tissue was collected for phosphopeptide enrichment
        
        Again, we performed a mass spectrometry–based unbiased phosphoproteomic screen
  - - - investigated the morphology of mitochondria upon sensory food perception and refeeding by performing transmission electron microscopy
        
        Livers were subjected to TEM and hepatic mitochondrial area, perimeter, aspect ratio (ratio between mitochondrial length and width), and number of mitochondria per image were analyzed in a blinded manner.
        
        next asked whether optogenetic activation of hypothalamic POMC neurons could recapitulate these alterations
        
        POMC Cre mice were stereotactically injected with an adeno-associated virus (AAV), allowing for Cre-dependent expression of either enhanced yellow fluorescent protein (EYFP) as control or the light-activatable ChR2.
  - - - To define the kinase responsible for phosphorylation of S131 in MFF, we used publicly available kinase prediction tools of eukaryotic datasets.
        
        Serine 131, as well as the surrounding amino acids of MFFS131, is well conserved among higher species
        
        We next aimed to validate whether MFFS131 indeed serves as a direct substrate of AKT.
        
        purified bacterially expressed murine MFF protein and used mass spectrometry to detect phosphopeptides in an in vitro kinase assay
        
        used a known AKT substrate (AKT/SKG substrate) in parallel reactions
        
        To further validate whether AKT kinase is activated during food perception, refeeding, and POMC neuron activation, we investigated the presence of AKT kinase substrate motifs [RXRXX(S/T)] within all regulated phosphosites in the previous phosphoproteomic datasets
        
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  - - - generated two knock-in mouse models by CRISPR-Cas9 gene editing
        
        one mouse line in which MFFS131 cannot be phosphorylated
        
        MFFS131G mice carry a genomic mutation that leads to the substitution of serine 131 of MFF with glycine
        
        isolated MFFS131G and MFFS131D mouse embryonic fibroblasts (MEFs) and primary hepatocytes from these mice, to study in vitro the consequences of altering the S131 phosphorylation site
        
        compared the ability of AKT activation by insulin stimulation in immortalized control compared to MFFS131G and MFFS131D MEFs
        
        another line in which a constant phosphorylation of MFFS131 is mimicked
        
        MFFS131D mice carry a mutation that leads to the substitution of serine 131 of MFF with aspartate
    - - functional consequences of altered MFFS131 phosphorylation in vitro and in vivo.
        
        MFFS131G MEFs displayed higher oxidative phosphorylation
        
        MFFS131D MEFs displayed a lower mitochondrial respiration
        
        primary hepatocytes isolated from MFFS131G mice
        
        assessed mitochondrial cristae morphology in the liver of MFFS131G mice
        
        compared mitochondria–ER contact sites in the liver of MFFS131G and control mice.
        
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      - investigated whether MFFS131G mice display alterations in systemic glucose metabolism.
        
        Mice carrying a homozygous nonphosphorylatable MFFS131G mutation did not show any alterations in body weight, body composition, or glucose tolerance in comparison to controls
        
        but showed mild impairment of insulin sensitivity as evidenced in an insulin tolerance test