High glucose-induced mitochondrial respiration and reactive oxygen species in mouse cerebral pericytes is reversed by pharmacological inhibition of mitochondrial carbonic anhydrases: Implications for cerebral microvascular disease in diabetes

Highlights

• High glucose enhances respiration (metabolism of glucose) in cerebral pericytes.

• Enhanced respiration leads to overproduction of reactive oxygen species (ROS).

• ROS damage pericytes in microvasculature of insulin insensitive tissues.

• Inhibition of mitochondrial carbonic anhydrases (mCAs) slows respiration and ROS.

• Mitochondrial CAs are a potential target for microvascular disease in diabetes.

Abstract

Hyperglycemia-induced oxidative stress leads to diabetes-associated damage to the microvasculature of the brain. Pericytes in close proximity to endothelial cells in the brain microvessels are vital to the integrity of the blood–brain barrier and are especially susceptible to oxidative stress. According to our recently published results, streptozotocin-diabetic mouse brain exhibits oxidative stress and loose pericytes by twelve weeks of diabetes, and cerebral pericytes cultured in high glucose media suffer intracellular oxidative stress and apoptosis. Oxidative stress in diabetes is hypothesized to be caused by reactive oxygen species (ROS) produced during hyperglycemia-induced enhanced oxidative metabolism of glucose (respiration). To test this hypothesis, we investigated the effect of high glucose on respiration rate and ROS production in mouse cerebral pericytes. Previously, we showed that pharmacological inhibition of mitochondrial carbonic anhydrases protects the brain from oxidative stress and pericyte loss. The high glucose-induced intracellular oxidative stress and apoptosis of pericytes in culture were also reversed by inhibition of mitochondrial carbonic anhydrases. Therefore, we extended our current study to determine the effect of these inhibitors on high glucose-induced increases in pericyte respiration and ROS. We now report that both the respiration and ROS are significantly increased in pericytes challenged with high glucose. Furthermore, inhibition of mitochondrial carbonic anhydrases significantly slowed down both the rate of respiration and ROS production. These data provide new evidence that pharmacological inhibitors of mitochondrial carbonic anhydrases, already in clinical use, may prove beneficial in protecting the brain from oxidative stress caused by ROS produced as a consequence of hyperglycemia-induced enhanced respiration.

Introduction

Complications of diabetes in the microvasculature of the brain are caused by oxidative stress [2], [3], [20]. These complications include pericyte loss [20], disruption of the blood–brain barrier [1], [11], and cognitive decline [4], [15], [22], [28], [30], [32]. The blood–brain barrier is formed largely by endothelial cells in the microvasculature of the central nervous system. Cerebral pericytes, another type of cell in close proximity to endothelial cells, are vital for the integrity of the blood–brain barrier [1]. Pericytes are especially susceptible to oxidative stress, which leads to the cell death by apoptosis [25].

Oxidative stress in diabetes is caused by reactive oxygen species (ROS) [7], [18], [31], which are primarily generated during mitochondrial oxidative metabolism of glucose (respiration). These mitochondrial ROS activate other biochemical pathways such as polyol pathway [5], [19], advanced glycation end products (AGE) formation [8], [10], [16], protein kinase C activation [13], [33], and hexosamine pathway [17], [24], which in turn propagate more ROS. The pathological levels of ROS produced from these pathways cause oxidative stress, leading to diabetic complications in the brain. Previously, we and others [2], [3], [20], [25] hypothesized that a hyperglycemia-induced increase in the rate of respiration is responsible for the production of high levels of mitochondrial ROS in relatively insulin insensitive tissues such as the brain. However, there are no published reports to show an increase in the rate of respiration either in the diabetic brain in response to hyperglycemia or in cultured cerebral pericytes upon high glucose challenge.

In this study, we investigated the effect of high glucose on the rate of respiration and ROS production in cerebral pericytes in culture. In addition, we studied the effect of pharmacological inhibition of mitochondrial carbonic anhydrases on these high glucose-induced changes in cerebral pericytes.

Mitochondrial carbonic anhydrases VA and VB regulate the rate of respiration [20], [25]. Mice in which these enzymes have been genetically knocked out exhibit reduced oxidative stress in the brain [26]. Pharmacological inhibition of mitochondrial carbonic anhydrases also protects the mouse brain from oxidative stress and pericyte loss [20]. The high glucose-induced intracellular oxidative stress and apoptosis of pericytes are remedied by treatment with mitochondrial carbonic anhydrase inhibitors (CAIs) [25] as well.

We now report for the first time that the rate of respiration is significantly higher in cerebral pericytes challenged with high glucose compared to those in normal glucose. The amount of ROS is also significantly higher in these cells. Furthermore, we show a reversal in high glucose-induced rapid rate of respiration as well as the amount of ROS produced upon pharmacological inhibition of mitochondrial carbonic anhydrases.

These studies provide further evidence to recognize mitochondrial carbonic anhydrases as an important pathway that can be targeted therapeutically to prevent diabetic brain injury. Pharmacological inhibitors of mitochondrial carbonic anhydrases are in clinical use for a variety of disorders, including obesity [9], and can be safely tested for this new indication in translational research.