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Food perception promotes phosphorylation of MFFS131 and mitochondrial…
Food perception promotes phosphorylation of MFFS131 and mitochondrial fragmentation in liver
Introduction
Liver mitochondria
Predictive responses
the neuronal and molecular mechanisms underlying the regulation of anticipatory changes in liver upon fasting–refeeding transitions remain only partly defined.
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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Mitochondria
Mitochondria serve as nutrient sensors and are central to glucose and lipid metabolism in hepatocytes
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
Hypothesis
A1:Whether hepatic MFF as a target for phosphorylation upon sensory food perception, refeeding, and POMC neuron activation
A2: Whether hepatic mitochondrial morphology rapidly changes upon sensory food perception, refeeding, and POMC neuron activation
A3: How does MFF phosphorylation
A4: the relation between MFF,mitochondrial morphology and metabolic adaptations?
Results
Q1
Identification of hepatic MFF as a target for phosphorylation upon sensory food perception, refeeding, and POMC neuron activation
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
Q2
Hepatic mitochondrial morphology rapidly changes upon sensory food perception, refeeding, and POMC neuron activation
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.
Q3
Identification of AKT as a MFFpS131 kinase
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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Q4
AKT-dependent phosphorylation of MFFS131 promotes mitochondrial fragmentation in vitro and in vivo
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
Phosphorylation of MFFS131 is required for metabolic adaptations
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
优缺点
从中枢切入,查看短时摄食的改变
中枢仅观察了POMC,可在补充AgRP