Reduction in cardiolipin decreases mitochondrial spare respiratory capacity and increases glucose transport into and across human brain cerebral microvascular endothelial cells

Abstract

Microvessel endothelial cells form part of the blood-brain barrier, a restrictively permeable interface that allows transport of only specific compounds into the brain. Cardiolipin is a mitochondrial phospholipid required for function of the electron transport chain and ATP generation. We examined the role of cardiolipin in maintaining mitochondrial function necessary to support barrier properties of brain microvessel endothelial cells. Knockdown of the terminal enzyme of cardiolipin synthesis, cardiolipin synthase, in hCMEC/D3 cells resulted in decreased cellular cardiolipin levels compared to controls. The reduction in cardiolipin resulted in decreased mitochondrial spare respiratory capacity, increased pyruvate kinase activity, and increased 2-deoxy-[(3) H]glucose uptake and glucose transporter-1 expression and localization to membranes in hCMEC/D3 cells compared to controls. The mechanism for the increase in glucose uptake was an increase in adenosine-5'-monophosphate kinase and protein kinase B activity and decreased glycogen synthase kinase 3 beta activity. Knockdown of cardiolipin synthase did not affect permeability of fluorescent dextran across confluent hCMEC/D3 monolayers grown on Transwell(®) inserts. In contrast, knockdown of cardiolipin synthase resulted in an increase in 2-deoxy-[(3) H]glucose transport across these monolayers compared to controls. The data indicate that in hCMEC/D3 cells, spare respiratory capacity is dependent on cardiolipin. In addition, reduction in cardiolipin in these cells alters their cellular energy status and this results in increased glucose transport into and across hCMEC/D3 monolayers. Microvessel endothelial cells form part of the blood-brain barrier, a restrictively permeable interface that allows transport of only specific compounds into the brain. In human adult brain endothelial cell hCMEC/D3 monolayers cultured on Transwell(®) plates, knockdown of cardiolipin synthase results in decrease in mitochondrial cardiolipin and decreased mitochondrial spare respiratory capacity. The reduced cardiolipin results in an increased activity of adenosine monophosphate kinase (pAMPK) and protein kinase B (pAKT) and decreased activity of glycogen synthase kinase 3 beta (pGSK3β) which results in elevated glucose transporter-1 (GLUT-1) expression and association with membranes. This in turn increases 2-dexoyglucose uptake from the apical medium into the cells with a resultant 2-deoxyglucose movement into the basolateral medium.

Glucose Uptake and Triacylglycerol Synthesis Are Increased in Barth Syndrome Lymphoblasts

“…In addition, the presence of abundant glucose in the culture medium, combined with a minimal demand for "work", may mask the effects of Ptd 2 Gro loss on substrate utilization, fat accumulation, or glucose disposal. We recently showed that a reduction in Ptd 2 Gro in human brain endothelial HCMEC/ D3 cells led to increased glucose uptake and glycolysis [20]. In summary, we show that BTHS lymphoblasts exhibit increased glucose uptake and TAG synthesis from glucose.…”

Mitochondrial spare respiratory capacity: Mechanisms, regulation, and significance in non‐transformed and cancer cells

“…Thus, reduction in the cardiolipin content decreases SRC levels. 8 In line with this, mild uncoupling resulting in a moderate increase in proton permeability leading to a reduction in ROS production without affecting ATP levels could decrease SRC levels, probably by increasing basal respiration. 8,9…”

“…7 One should note that FCCP concentration needs further dose optimization studies. Optimal FCCP doses to obtain SRC depend on experimental parameters such as temperature 8 and excessive uncoupling promotes ROS-dependent cell death and eventually energetic crisis. 9 Because optimal doses can vary according to the different experimental conditions, it is better to do sequential additions of uncouplers to achieve maximal rate ( Figure 1).…”

The Key Roles of GSK-3β in Regulating Mitochondrial Activity

“…It has been demonstrated that cardiolipid synthase knockdown significantly decrease the capacity of spare respiration, but does not affect the activity of complex I in hCMEC/D3 cells. The possible compensatory mechanism might be associated with increased utilization of glucose, as shown by increased glycolysis and increased activity of pyruvate kinase and GLUT1, which are correlated with inactivation of GSK-3β [48]. GSK-3β inactivation or GSK-3β-Ser9 phosphorylation maintains the efficiency of oxidative phosphorylation (OXPHOS) and controls the production of ROS.…”