Detailed methodology can be found in the Supplementary Information . In short, tissue preparation, immunocytochemistry, synaptosomal respiration, Real-time PCR, quantitative and qualitative electron microscopic analyses of mitochondria and synapse number, electrophysiology and feeding behaviour analyses were performed as previously described1 (link)–3 (link),9 (link),23 (link),36 (link),39 (link).
The mitochondrial membrane potential was measured in isolated synaptosomal mitochondrial fractions from the hypothalamus using ΔΨm-sensitive TMRE and fluorescent spectrofluorophotometry. Non-esterified fatty acids were measured in plasma using a colorimetric non-esterified fatty acid (NEFA) kit (Wako) following the manufacturer’s instructions. LCFA CoAs were measured by mass spectroscopy.
ROS production was quantified using dichlorodihydrofluorescein diacetate (DCF) in mitochondrial fractions and using DHE in fixed tissue.
The mitochondrial membrane potential was measured in isolated synaptosomal mitochondrial fractions from the hypothalamus using ΔΨm-sensitive TMRE and fluorescent spectrofluorophotometry. Non-esterified fatty acids were measured in plasma using a colorimetric non-esterified fatty acid (NEFA) kit (Wako) following the manufacturer’s instructions. LCFA CoAs were measured by mass spectroscopy.
ROS production was quantified using dichlorodihydrofluorescein diacetate (DCF) in mitochondrial fractions and using DHE in fixed tissue.
