Background
In recent years, cigarette smoking has been considered the second-leading risk factor for death and, in the United States, it increases the risk of stroke by 2–4 fold [
1]. In addition to cigarette smoking, electronic nicotine vaping device or “e-cigarette” usage has increased in recent years. A recent study in Addiction reports that e-cigarette usage is growing in both populations of former smokers or current smokers as an aid to cut down or quit smoking [
2]. Even though it is known that e-cigarettes may provide a healthier option compared to tobacco cigarettes with respect to carcinogens, the effects of long-term exposure to sometimes variable doses of nicotine from e-cigarettes is yet to be determined and will need to be validated with longitudinal studies. Additionally, stroke has become a fourth leading cause of death and disability in the US [
1]. Occlusion of a major cerebral artery by an embolus or thrombosis can result in transient or permanent deprivation of nutrient and oxygen supply to parts of the brain. The brain relies heavily on a continuous glucose supply that is regulated across the blood–brain barrier (BBB) via glucose transporters to provide the fuel to maintain cellular ATP as an energy source for brain activity [
3-
5]. Subsequent deficiency in the major obligatory brain fuels, glucose and oxygen, elicits a number of important neurochemical mechanisms (e.g. excitotoxicity, oxidative stress and inflammation) which can lead to irreversible brain damage. This changing brain microenvironment is tightly regulated by the brain microvasculature which functions to segregate the blood from brain interstitial fluid. The endothelial cells of the BBB provide a dynamic interface between the blood and central nervous system (CNS), maintaining brain homeostasis by selectively limiting the passage of solutes/nutrient/ions from the circulating blood into and out of the brain and it plays an important role in determining the fate of brain tissue after stroke. Brain microvascular endothelial cells working in concert with astrocytes, pericytes and neurons form a neurovascular unit (NVU). Many of these key solutes and nutrients enter the brain by transcellular diffusion and others through passive or active carriers that may utilize receptor-mediated endocytosis [
3].
In stroke, loss of blood supply increases energy demand causing the nutrient and ion transporter activity to adapt to deprived conditions. Any changes in the BBB dynamic function resulting from altered function/expression of solute/ion transporters can worsen brain pathophysiology in a number of neurological diseases and disorders including stroke [
6,
3]. Cerebral glucose transport and metabolic derangement during ischemia have been observed in both animals [
7] and human studies [
8]. An initial increase in glucose metabolism/utilization occurs due to release of excitatory amino acids in response to ischemic insults, followed by significant reduction in glucose metabolism in the same brain regions with consequent increase in function and expression of glucose transporters at the BBB and in brain [
9,
10]. Enhanced glucose transporter levels have been suggested to compensate for the lack of glucose availability to the brain in ischemic conditions. Several mechanisms have been suggested to substantiate the adaptive increases in the glucose transporter expression during IR injury. It has been shown that the regulation of GLUT1expression in ischemic brain endothelial cells can occur through a activation of phosphoinositide-3 kinase (PI3K)/Akt pathway via vascular endothelial growth factor [
11], HIF1α activation [
12] etc.
Changes in BBB function due to nicotine and the components of tobacco smoke can also have significant effects on brain injury [
13-
16]. The observed effects of chronic nicotine exposure on brain and BBB function are seemingly mostly detrimental during stroke. Nicotine has been reported to cause changes in BBB function that include alterations in expression or function of BBB-associated proteins [
17,
14], cerebrovascular blood flow [
18], BBB permeability [
19,
13], increases in cerebrovascular thrombosis [
20] and an increased post-ischemic inflammatory response [
15]. Interestingly, both acute and chronic administration of nicotine has been shown to decrease glucose transport rates across the BBB but increase global glucose utilization in normal rat brain [
21-
24]. Duelli et al., (1998), have reported decreased 3-O-[14C] methylglucose transfer across the BBB in rats infused with nicotine for 1 week. Those investigators suggested that an increase in local cerebral glucose utilization and reduced glucose transfer rate is associated with an increase in GLUT1 glucose transporter in rats pre-exposed to nicotine for one week [
22,
23]. Those studies utilized an
in vivo method to estimate glucose influx and efflux rate constants by injecting the radiotracer IV and analyzing radiotracer amount in blood and brain of animals at set time points. Similar reports in humans are sparse and have demonstrated an overall small reduction in global cerebral glucose utilization although several brain regions showed relative enhanced glucose utilization [
25,
26]. Moreover, none of the existing studies have investigated the effects of nicotine on glucose transport rate and expression at the BBB during stroke. Thus, to identify an effect of nicotine on BBB glucose transport rate during stroke, we focused our investigations on the validation of these previous findings by utilizing the
in situ brain perfusion method. This allowed estimation of the initial rate of glucose influx across the BBB under equilibrium conditions with no systemic interference [
27]. Further, we also studied the nicotine-induced changes in expression of GLUT1 during stroke to help explain some of the possible nicotine and/or smoking-related changes in cerebrovascular functions in both normal and ischemic brain. Specifically, our studies evaluated BBB glucose transporter function and expression during ischemic stroke in nicotine pre-exposed animals.
Discussion
In our studies, we specifically determined unidirectional glucose transfer kinetics using an
in situ brain perfusion method. In that method, we can precisely study and control substrate influx across the BBB since the perfusion medium is infused with a known concentration of glucose, at a controlled infusion rate, for a defined interval of time, without any systemic interference as seen with single intra-arterial injection in intact animals. Previously, other reports have shown that both acute and chronic administration of nicotine decreases glucose transport but increases glucose utilization in normal rat brain [
22,
23]. Similar to previously published glucose transporter kinetics, our data further reinforces that nicotine reduces glucose transfer rates across the BBB with no change or a slight increase in total vascular glucose transporter densities in nicotine-exposed sham animals. The rate of D-glucose influx in saline-infused sham animals is also in agreement with values for unidirectional blood–brain glucose transfer obtained in our laboratory [
3] and by others in rats [
23,
32,
33]. Our study further confirms enhanced GLUT1 transport influx rates and expression in ischemic mice compared to saline-infused sham animals [
34]. Interestingly, in our studies, 14 day nicotine exposure before tMCAO reduced the ischemia-enhanced glucose transport rate across the BBB. Chronic nicotine exposure has been shown to affect multiple transporter systems at the BBB and in brain in physiological and pathophysiological conditions. Reduced expression of Na, K-ATPase at the BBB by chronic nicotine administration has been demonstrated to increase focal cerebral ischemic injury in rats [
17] and a similar trend was observed in cultured brain endothelial cells exposed to hypoxia/aglycemia with or without nicotine and cotinine [
14]. Nicotine has been shown to dose-dependently inhibit the increased NKCC activity observed during hypoxia/aglycemia in
in vitro bovine brain microvascular cells [
14]. Moreover, nicotine exposure increased both edema and infarct volume and worsened neurobehavioural outcomes in a 24 h permanent MCAO mouse model [
16]. Importantly, the availability of glucose during and after stroke dramatically affects ischemic outcome [
35-
38]. Numerous studies examining different glucose-lowering strategies in patients with pre and post-ischemic hyperglycemia have been summarized in a recent review [
39]. These studies indicate that tight glucose control is associated with a major risk of severe symptomatic and asymptomatic hypoglycemic episodes which further worsens the stroke injury [
40-
45]. Perhaps, the extension of observed reduced glucose transport in nicotine pre-exposed animals, across the BBB under normal conditions and during ischemic reperfusion may create a more glucose deprived state at the NVU and an exaggeration of ischemia-induced brain injury.
In this study, the direct mechanisms underlying nicotine-induced changes in GLUT1 transporter function or expression were not determined and they will be the focus of future studies. However, several mechanistic investigations from our lab have shown that nicotine can inhibit hypoxia-induced increased NKCC activity via a PKC mediated phosphorylation pathway [
46]. We have also demonstrated an inhibitory effect of nicotine on several PKC isoforms during hypoxic conditions in bovine brain microvascular endothelial cells [
46]. Similarly, other
in vitro studies have indicated PKC-mediated glucose transporter activity in a variety of different tissues [
47] and retinal capillaries [
48]. Specifically in endothelial cells, PKC mediated increased glucose transport via translocation modulation has been demonstrated [
49,
50]. Likewise, in human brain, PKC-mediated GLUT1 expression has been suggested based on simultaneous changes in PKC co-localization with changes in GLUT1 density within endothelial domains [
51]. Thus, we speculate nicotine-induced PKC changes could alter GLUT1 translocation in endothelial cells and may explain the possible reduced glucose transfer rate in the presence of no overall change or a slight increase in glucose transporter densities in brain capillary endothelial cells as observed in our nicotine-infused sham animals.
It is important to note that nicotine administered at 4.5 mg/kg/day for 1 day, 1 week, and 2 weeks through Alzet minipumps, results in plasma levels of nicotine of 80–100 ng/mL and the major metabolite cotinine of >250 ng/mL, similar to a heavy smoker [
16]. A study by Abbruscato, et al., (2002) has previously demonstrated the direct dose-dependent effects of nicotine and cotinine on increased paracellular BBB permeability and reduced tight junctional protein expression, using bovine brain microvascular endothelial cells in culture, an
in vitro BBB model. [
13]. Also, others have demonstrated that plasma levels of nicotine and cotinine equivalent to smokers causes an increase in paracellular permeability in rats when exposed acutely (12 hours) [
52]. In contrast, other studies have shown that a chronic nicotine dose (28 days) of 4.5 mg/kg/day does not affect the BBB integrity measured by sucrose [
53]. Similar to previous reports, our results also suggest no change in the BBB integrity in nicotine pre-exposed animals. We calculated a glucose transport rate (
K
in
) that was corrected for vascular space measured simultaneously in the same animals. We confirmed that the observed reduced glucose transport rate in ischemic animals was due to down-regulation of GLUT1 proteins in brain endothelial cells and not to changes in vascular volume, further validating the observed effect of nicotine on glucose transporter densities in brain [
22]. Further, 4.5 mg/kg/day of nicotine does not affect cerebral blood flow as measured with
in situ brain perfusion using radiotracer diazepam [
54]. Moreover, we believe that glucose influx
K
in
is independent of blood flow since estimated initial influx rate is relatively small compared to estimated cerebral blood flow in the mouse under similar conditions.
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Competing interests
The authors declare no financial or non-financial competing interests.
Authors’ contributions
KKS carried out the control experiments, conceived of the study and, participated in the design, acquisition, analysis, and interpretation of data and drafting of manuscript. PRB carried out experiments, participated in design and acquisition of data. TJA conceived of the study and participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.
Thomas J Abbruscato, Ph.D: Professor and Chair Pharmaceutical Sciences, Associate Dean, Graduate School of Biomedical Sciences, Texas Tech University HSC, School of Pharmacy.
Kaushik K Shah, Ph.D: Research Associate, Texas Tech University HSC, School of Pharmacy.
Purushotham Reddy Boreddy, Ph.D: Current Affiliation: Research Associate, National Centre for Cell Science (NCCS), Cancer Biology; Past Affiliation: Post-Doctorate Research Fellow, Texas Tech University HSC, School of Pharmacy.