Extracellular citrate impacts hypothalamic mitochondrial function and activates AMPK

 

Citrate, one of the most abundant metabolites in the cerebrospinal fluid (CSF), is a potent chelator and important for neuronal activity. Decreased blood citrate levels were found in diabetic humans. Global deletion of the citrate transporter SLC13A5 increases plasma citrate levels, activates hepatic AMP-activated protein kinase (AMPK) and protects against obesity and insulin resistance, suggesting that increased extracellular citrate levels might be beneficial. Yet, the role of extracellular citrate on brain function is not well understood and was investigated in the current study.

Citric acid content in CSF and plasma samples of db/db mice was determined using a metabolomics approach. To delineate the effect of altered citrate concentration, we performed in vitro and ex vivo stimulations with 1mM citrate and analyzed cellular metabolism and divalent cations concentration using western blot and qPCR techniques, mitochondrial activity assessment as well as ICP mass spectrometry.

We demonstrate that iso-/citrate is significantly reduced in CSF and plasma of db/db compared to db/+ mice. Stimulation with 1mM citrate did not affect divalent cations concentration nor insulin signaling, but lowered basal respiration by 19,7% and ATP production by 37,7% only in neurons without inducing oxidative stress exemplified by unaltered protein carbonylation, mitochondrial stress or neuroinflammatory markers such as IL-6 and TNF-α. As a consequence, citrate causes AMPK activation in neurons but surprisingly not in glia and increases AMPK activity in hypothalamic brain slices.

Our data point to a novel regulatory role of extracellular citrate on neuronal metabolism by decreasing basal respiration and activating hypothalamic AMPK.