Effect of High Glucose Condition on Glucose Metabolism in Primary Astrocytes

Abstract

In the brain, glucose enters astrocytes through glucose transporter (GLUT1) and either enters glycolysis or the glycogen shunt. Astrocytes meet the energy needs of neurons by building up and breaking down their glycogen supply. High glucose exposure causes astrocyte dysregulation, but its effects on glucose metabolism are relatively unknown. We hypothesized that high glucose conditioning induces a glycogenic state in the astrocyte, resulting in an inefficient mobilization of substrates when challenged with glucose deprivation. Using neonatal rat astrocytes, we used normal glucose (NG, 5.5mM) vs. high glucose (HG, 25mM) feeding media and measured cell membrane GLUT1 expression, glucose analog uptake, glycogen content, and cellular bioenergetics. This study demonstrates that HG conditioning causes increased glucose analog uptake (p<0.05) without affecting GLUT1 membrane expression when compared to NG conditioned astrocytes. Increased glucose uptake in HG astrocytes is associated with higher baseline glycogen content compared to NG exposed astrocytes (p<0.05). When challenged with glucose deprivation, HG astrocytes break down more than double the amount of glycogen molecules compared to NG astrocytes, although they break down a similar percentage of the starting glycogen stores (NG=62%, HG=55%). Additionally, HG conditioning negatively impacts astrocyte maximal respiration and glycolytic reserve capacity assessed by the Seahorse mitochondrial stress test and glycolytic stress test, respectively (p<0.05). These results suggest that HG conditioning shifts astrocytes towards glycogen storage at baseline. Despite increased glycogen storage, HG astrocytes demonstrate decreased metabolic efficiency and capacity putting them at higher risk during extended periods of glucose deprivation.

1. Introduction

Hyperglycemia complicates conditions in several disease states, such as diabetes mellitus. The high glucose condition resulting from diabetes alters the functioning of brain cells and leads to neurological impairment (1). The astrocyte is the key cell responding to changes in glucose in the brain microenvironment and alters its handling of substrate according to supply of glucose. The primary mechanism leading to pathophysiological complications associated with diabetes is glucose uptake and subsequent metabolism. It is therefore imperative to study the effects of high glucose exposure on astrocyte glucose metabolism in order to understand the pathogenesis of the complications related to the diabetic brain (2).

Glucose metabolism has various effects on brain functioning, especially the relationship between astrocytes and neurons (3). Astrocytes play a critical role in brain metabolism by providing metabolic substrate in response to increased energy needs of neurons. Glucose is the primary energy source of the brain and is allowed into the cells via facilitative glucose transporters, GLUTs. The GLUT1 isoform is primarily responsible for basal glucose uptake by the astrocyte (4). The amount of glucose uptake depends on extracellular glucose availability (5). Once glucose enters the astrocyte, it is converted to glucose-6-phosphate (G6P) and either enters the glycogen shunt or glycolysis. In the glycogen shunt, glycogenic enzymes such as glycogen synthase build up glycogen stores. When the astrocyte or surrounding neurons require energy metabolites, glycogen phosphorylase and debranching enzyme break down glycogen to G6P in order to perform glycolysis (5).

In times of glucose availability, the astrocyte stores glycogen for future use when neuronal energy need increases. However, when challenged with glucose deficiency, glycogen supplies are used to generate lactate, which is then shuttled to nearby neurons. Similarly, during times of active neural firing, when energy demand is high, astrocytes release lactate. This system in which neurons rely on astrocytic energy reservoirs in order to function is termed the Astrocyte-Neuron-Lactate-Shuttle (ANLS). It is believed that glycogen from astrocytes functions as a protective mechanism against hypoglycemia, ensuring preservation of neuronal function (3).

Instability of glucose levels in the astrocytic microenvironment impairs astrocyte metabolism and brain homeostasis. It is understood that fluctuating glucose levels induces glial injury by decreasing astrocyte cell proliferation, inducing mitochondrial dysfunction, increasing oxidative stress, impairing glutamate metabolism and increasing pro-inflammatory cytokine release. High glucose conditioned astrocytes have a more exaggerated production of reactive oxygen species than normal glucose cells when challenged with glucose deprivation (5). Similarly, high glucose conditions have been found to induce astrocyte reactivity, which promotes oxidative damage, in the hippocampus of type 1 diabetic mice and C6 cells (7,8). These reactions by astrocytes may be the earliest response of the brain to fluctuations in microenvironmental glucose levels and likely underlie diabetes-related brain impairment (5). As astrocytes provide necessary energy for neuronal function, an understanding of glucose handling by astrocytes would inform how this impairment takes place in such disease states. After uptake, some glucose molecules are shunted through glycogen before reentering glycolysis, a pathway known as the glycogen shunt. The significance of glycogen in astrocyte metabolism is exhibited by high activity of the glycogen shunt as well as the finding that inhibition of glycogen degradation leads to a disproportional increase in glycolysis (9). It has previously been shown that exposure of astrocytes to high glucose media (25mM) leads to increased glycogen storage and ATP (10) reinforcing the idea that glycogen synthesis is a luxury that is affordable in times of ample energy supply (11). However, the response of these conditioned cells to glucose deprivation has not been fully elucidated. Our study fills this gap by investigating how high glucose conditioning affects glycogen levels when challenged with a lack of environmental glucose, and tests the hypothesis that high glucose conditioning adversely affects the metabolic profile of astrocytes.

2. Results

2.1. High glucose causes increased astrocytic glucose uptake despite unchanged GLUT1 membrane expression

We used the Fluorometric Glucose Analog Uptake Assay to compare uptake of glucose analog in normal and high glucose conditioned astrocytes as a proxy for glucose uptake. At the end of a 1-hour exposure to the fluorescent glucose analog, 2-NBDG (2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino]-2-deoxy-d-glucose), in glucose-free media, HG conditioned astrocytes took up more intracellular 2-NBDG than NG astrocytes. This is evidenced by the significantly higher Normalized Fluorescent Index (NFI) in HG astrocytes compared to NG (Fig. 1A). Despite this increased glucose analog uptake, HG treatment did not alter GLUT1 expression at the cell membrane (Fig. 1B-C). GLUT1 membrane expression was evaluated by subcellular fractionation of primary astrocyte cultures to isolate the membrane fraction, and subsequent Western blot analysis. This data indicates that astrocytic GLUT1 membrane expression is not affected by glucose concentration in the environment, although these transporters may have an increased capacity for glucose uptake.

Fig. 1.

Effect of high glucose conditioning on glucose uptake. (A) Normalized Fluorescent Index (NFI) normalized to total protein (n=4). (B) Expression of GLUT1 in the membrane fraction over total protein (n=4). (C) Representative Western bots of GLUT1 and total protein (TP) (strongest band at 50kDa). Individual data points are plotted, mean is shown, and error bars indicate standard deviations. ANOVA (Tukey’s multiple comparisons test) # P<0.05.

2.2. High glucose shifts astrocytes towards glycogen storage

We used the Fluorometric Glycogen Assay to evaluate baseline glycogen levels in normal and high glucose astrocytes. Astrocytes exposed to HG conditions have significantly increased glycogen stores at baseline as compared to astrocytes exposed to NG conditions (Fig. 2). This shows that when more glucose is available in the extracellular environment, astrocytes can build up their glycogen stores accordingly.

Fig. 2.

Effect of high glucose conditioning on glycogen generation. Glycogen content normalized to total protein (n=5). Individual data points are plotted, mean is shown, and error bars indicate standard deviations. Student’s t-test: + P<0.05

2.3. High glucose does not affect the ability of astrocytes to break down glycogen

The Fluorometric Glycogen Assay was also used to evaluate glycogen levels in response to unavailability of glucose. After a 1-hour glucose deprivation, both NG and HG exposed astrocytes successfully mobilized glycogen stores (Fig. 3). After glucose deprivation, NG and HG astrocytes depleted a similar percentage of their original glycogen stores (62% for NG, 55% for HG), although HG astrocytes broke down more than twice the amount of absolute glycogen compared to NG astrocytes (3.755 ng/mg for NG and 8.556 ng/mg for HG). This data suggests that in times of low glucose availability or high metabolic demand, high glucose astrocytes break down a disproportionate amount of glycogen compared to normal glucose astrocytes.

Fig. 3.

Glycogen breakdown when challenged with glucose deprivation. Glycogen content normalized to total protein in control conditions (NG, HG, n=5) and after a 1-hour glucose deprivation challenge (NG 0G, HG 0G, n=6). Individual data points are plotted, mean is shown, and error bars indicate standard deviations. Student’s t-test: ++ P<0.01, ANOVA (Sidak’s multiple comparisons test) ## P<0.01.

2.4. High glucose reduces astrocyte metabolic capacity

We used the Seahorse XF assay to determine the bioenergetic responses of astrocytes cultured in NG and HG conditions. Astrocytes were subjected to the cell mito stress test to determine parameters of mitochondrial function measured as Oxygen Consumption Rate (OCR) (Fig. 4A-B). The final reaction in the electron transport chain, oxygen consumption, is an indirect measurement of mitochondrial function. OCR is both a measurement for the flux of electrons through the respiratory chain, as well as of the processes that consume energy (12). Conditioning astrocytes in HG media did not alter baseline respiration or ATP production (Fig. 4C-D). Application of Carbonyl cyanide-4 (trifluoromethoxy) phenylhydrazone (FCCP) to stimulate uninhibited flow through the electron transport chain did reveal that HG exposure significantly reduces maximal oxygen consumption rate, spare respiratory capacity, as well as coupling efficiency (Fig. 4E-G). Further, the glycolysis stress test was used to assess parameters of glycolytic flux after a one-hour glucose deprivation measured as the Extracellular Acidification Rate (ECAR) (Fig. 5A-E). Extracellular acidification rate of the surrounding media, which is essentially a measurement of pH, is determined based on the excretion of lactic acid per unit time after its conversion from pyruvate (13). Reintroducing a saturable amount of glucose after deprivation demonstrated decreased glycolysis in the HG compared to NG astrocytes (Fig. 5C). Shutting down oxidative phosphorylation with oligomycin further demonstrated that HG conditioning reduces astrocyte glycolytic capacity and glycolytic reserve (Fig. 5D-E).

Fig. 4.

HG conditioning causes decreased astrocyte maximal respiration compared to NG astrocytes, as assessed by the Seahorse mitochondrial stress test. Astrocytes were subjected to the cell mito stress test to determine parameters of mitochondrial function measured as Oxygen Consumption Rate (OCR) (A-B). HG did not alter baseline respiration or ATP production (C-D). HG exposure significantly reduces maximal oxygen consumption rate, spare respiratory capacity, as well as coupling efficiency (E-G). Student’s t-test: * P<0.05.

Fig. 5.

HG conditioning causes decreased glycolytic reserve capacity compared to NG astrocytes, as assessed by the Seahorse glycolytic stress test. The glycolysis stress test was used to assess parameters of glycolytic flux after a one-hour glucose deprivation measured as the Extracellular Acidification Rate (ECAR) (A-B). There was decreased glycolysis (C), glycolytic capacity (D) and glycolytic reserve (E) in HG compared to NG. Student’s t-test: * P<0.05.

3. Discussion

High glucose conditions, which are manifested in disease states such as diabetes, cause cerebral dysfunction down to the cellular level. Astrocytes serve a critical role in the brain as glucose sensors, energy reservoirs, and providers of energy substrates to neurons. Alterations to astrocytic glucose metabolism could have profound effects on the ability of these cells to meet metabolic needs of neurons in the microenvironment. The effects of high glucose on astrocytic glucose metabolism has not been completely elucidated to date. The novel findings of this study, which include that high glucose conditioning of astrocytes causes increased glucose uptake, increased glycogen storage, but reductions in maximal mitochondrial respiration and glycolytic capacity, add to our understanding of astrocytic glucose metabolism in the setting of high glucose.

In the present work using an in vitro model of high glucose, we demonstrated that when exposed to excess glucose, astrocytes do not alter GLUT1 membrane expression. Previously, an in vivo model of short-term fasting in adult rats by Dakic et al. demonstrated that hypothalamic endothelial cells and neurons upregulate membrane GLUT1 and GLUT3, respectively, in response to glucose deprivation, whereas astrocytes showed no change in GLUT1 membrane expression (6). Together, these findings suggest that astrocytic GLUT1 is not affected by extracellular glucose concentration. Despite similar GLUT1 membrane expression, we did observe an increased glucose uptake in astrocytes grown in HG conditions through the use of glucose analog 2-NBDG. Glucose is thought to be taken up by cell membrane GLUT1 via facilitated diffusion as the primary mechanism. Because this system relies on a gradient to function, one might expect that less glucose uptake would occur as intracellular glucose levels increase. However, as long as the glycogen shunt and/or glycolysis pathways continue to utilize this intracellular glucose, the gradient can be maintained. Glycogen synthase is a glucose-6-phosphate sensor (15) and glucose allosterically activates glycogen synthase to drive the glycogen shunt and thus help to maintain this gradient. The increased glucose analog uptake that we observed could alternatively be explained by the trans-acceleration property of GLUT1 - where unidirectional uptake of glucose is stimulated by the presence of intracellular sugar (7). Therefore, trans-acceleration in high glucose states may provide a metabolic advantage to the astrocyte, allowing rapid equilibration of intracellular and extracellular glucose levels and subsequent stimulation of glycogenesis.

To further investigate if the glycogen shunt or glycolytic activity increased to explain the increased glucose uptake, we first determined the amount of cellular glycogen in NG and HG astrocytes. Here we showed that glycogen content at baseline is increased in HG conditions compared to NG conditions. This increased glycogen content suggests that the glycogen shunt via glycogen synthase activity is increased in high glucose conditioning. This could be explained by the observed increased glucose uptake in HG conditions. As stated above, glucose itself acts as an allosteric activator of GS and inhibitor of GP. If HG astrocytes have more intracellular glucose compared to NG astrocytes, then this substrate could account for the increased glycogen storage at baseline. It is not known what effects high glucose treatment has on glycogen shunt enzymes such as GSK3P, AMPK, and PhK. This aligns with the discussion brought forth in the 2013 review article by DiNuzzo et al., which states that reciprocal regulation of GS and GP by AMPK does not render mutually exclusive synthesis and degradation of brain glycogen (8). As demonstrated by Oz et al., simultaneously active GS and GP account for steady-state glycogen turnover (9). However, the rate of glycogen turnover at steady-state is not zero due to allosteric effectors such as glucose and glucose-6- phosphate. At the microenvironmental level, individual glycogen molecules can be found in different states of synthesis and degradation. Further, we found that when HG astrocytes are challenged with a starvation period, astrocytes break down glycogen stores to a greater degree than NG astrocytes, likely to satisfy cellular metabolic needs. Therefore, HG astrocytes behave in a more inefficient manner compared to NG astrocytes in the starvation state, in addition to having a discrepancy in glycogen levels at baseline.

Next, we determined if HG conditioning alters astrocyte bioenergetics using the Seahorse assay to measure mitochondrial respiration and glycolytic flux. In our study, high glucose conditions reduced maximal mitochondrial respiration and measured parameters of glycolysis. Despite these responses to conditions of metabolic stress we did not observe changes in basal respiration or ATP production suggesting that astrocytes can adapt to chronic changes in glucose availability in the absence of stressors. The increased glycogen storage in HG likely is part of this adaptation allowing astrocytes to maintain intracellular gradients in order to normalize glucose uptake and basal respiration.

Spare respiratory capacity is a measure of the ability of the cell to respond to increased energy demand or stress (19). Mitochondrial respiration provides energy substrate for the astrocyte in times of increased energy need. Astrocytes derive energy from both glycolytic and oxidative pathways, but respiration has a higher energy yield as it produces the most ATP. Astrocytic filopodial and lamellipodial extensions, which account for 80% of their surface area, are too narrow to accommodate mitochondria. In these regions of the astrocyte, there is a dependence on glycolysis, glycogenolysis, and likely ATP from mitochondrial metabolism to satisfy energy demands (20). One of the novel findings from this study is that there is reduced astrocytic mitochondrial capacity and efficiency in high glucose conditions. This suggests that under stress, the astrocyte is rendered unable to sustain energy levels for cell maintenance of critical extensions that interact with other constituents of the neurovascular unit.

Conversion of glucose to glycogen in astrocytes provides a limited reserve of energy which may be important for sustaining neuronal function in periods of intense energy use. Neurons are the principal consumers of brain energy. The astrocyte-neuron lactate shuttle (ANLS) hypothesis proposes a possible mechanism by which neuronal activity could be linked to energy supply as described by Mason et al (10). More recent studies have identified roles for astrocytic glycogen in supporting memory consolidation and modulating glucose uptake in neurons (22). When neurons are active and releasing glutamate, uptake of glutamate into astrocytes and its subsequent conversion to glutamine are postulated to trigger glycolysis in the astrocyte, generating lactate that can be exported to neurons to be used as fuel. Our study indicates that astrocytes in high glucose environments at baseline have reduced glycolysis. When mitochondrial respiration is blocked by oligomycin, the glycolytic capacity of astrocytes in high glucose conditions is compromised. We speculate that because glycolytic capacity is reduced in high glucose conditions, when there is increased neuronal demand the astrocytes would be unable to shuttle adequate lactate to fuel neurons. Further studies are necessary to delineate the effect of high glucose on the communication and metabolic synergy of astrocytes and neurons.

4. Conclusions

Increased availability of glucose in the environment shifts astrocytes towards glycogen storage. While GLUT1 membrane expression remains stable, increased glucose uptake in HG conditions stimulates glycogenesis. Despite increased glycogen storage, high glucose exposed astrocytes break down glycogen when challenged with glucose deprivation. Additionally, HG conditioned astrocytes have a reduced ability to respond to metabolic stressors as evidenced by decreased glycolytic activity and reserve as well as decreased oxygen consumption rate and respiratory capacity compared to NG astrocytes. This suggests that when exposed to high glucose conditions, astrocytes capitalize on the available glucose and store more glycogen, although they are unable to efficiently utilize the energy via glycolysis or oxidative metabolism.

5. Materials and Methods

5.1. Animal Care

The animal protocols used in this study were approved by the Medical College of Wisconsin Institutional Animal Care and Use Committee. All rats had free access to a standard rat chow diet and drinking water. Rats were housed in a temperature (24 ± 2 °C), humidity (60 ± 10%), and 12-hour light cycle (lights on: 0600–1800) controlled environment.

5.2. Cell Culture

Sprague Dawley rat pups of 3 days of age were anesthetized with diethyl-ether, decapitated and the brain removed for preparation of astrocyte cultures as previously described (11). Briefly, the brain was dissected free of meninges, and the hippocampus was isolated and cut into small pieces, and transferred to sterile dish containing 20 U/ml papain (Worthington Biochemical Corp) and cysteine (0.15 mg/ml; Sigma) dissolved in Earle’s balanced salt solution (Gibco BRL) and incubated at 37 °C for 40 min with gentle agitation. Digestion was stopped by washing three times with an astrocyte growth medium containing Dulbecco’s Modified Eagle Medium (DMEM) (Thermo Fisher), 10% fetal bovine serum (FBS), 25 units/ml penicillin, 25 μg/ml streptomycin and 0.1% gentamicin (Invitrogen, Carlsbad, CA). The tissue was then dissociated by trituration with flame-narrowed Pasteur pipette and cell suspension was diluted with feeding medium and seeded at an initial density of approximately 2 * 10⁵ cells per square centimeter. For each litter of pups, which included both males and females, cells from the entire litter were combined and the cell mixture was subsequently distributed across culture plates. The cells were incubated at 37 °C in a 95%/5% mixture of atmospheric air and CO2. Cells were cultured in normal glucose DMEM (NG, 5.5mM) or high glucose DMEM (HG, 25mM) for 3–4 weeks over 3–4 passages. The medium was changed after 2 days and subsequently 2–3 times a week. Confluent monolayers of brain astrocytes were studied.

5.3. Whole Cell Lysate

Primary astrocyte cultures were rinsed twice with DPBS (Dulbecco’s Phosphate Buffered Saline) and lysed on ice with RIPA buffer (150 mM NaCl, 1.0% NP-40, 0.5% deoxycholate, 1 mM EDTA, 50 mM Tris pH 8.0) containing protease inhibitor cocktail (PharMingen, San Diego, CA, USA) composed of benzamidine HCl (16 μg/ml), phenanthroline (10 μg/ml), aprotinin (10 μg/ml), leupeptin (10 μg/ml), pepstatin A (10 μg/ml) and 50 mM PMSF. Samples were sonicated and centrifuged at 400g for 5 minutes. The supernatant was collected, and the protein concentration was quantified using the Bio-Rad protein assay (Bio-Rad Laboratories, Richmond, CA).

5.4. Subcellular Fractionation

Subcellular fractionation was performed to isolate the plasma membrane fraction of primary astrocytes using the Subcellular Protein Fractionation Kit for Cultured Cells (Thermo Scientific, Waltham, MA) according to manufacturer’s instructions. In brief, confluent primary astrocyte 100mm cell culture plates were rinsed twice with DPBS (Dulbecco’s Phosphate Buffered Saline). Cells were gently scraped from the plates with 1mL of cold DPBS per plate, and 3 plates of cells were combined into one 15mL Falcon tube. Tubes were centrifuged at 500 x g for 3 minutes. Supernatant was carefully removed with a pipet, and pellets were allowed to dry for 15 minutes by resting the tubes upside-down on a paper towel. Ice-cold Cytoplasmic Extraction Buffer with protease inhibitors were then added according to the approximate pellet size. After a 10 minute incubation at 4 degrees Celsius with gentle shaking, samples were centrifuged at 500 x g for 5 minutes. Supernatants (cytoplasmic extract) were removed. Ice-cold Membrane Extraction Buffer with protease inhibitors were added to the pellets and samples were vortexed briefly to mix. Tubes were again incubated at 4 degrees Celsius for 30 minutes with gentle shaking. Samples were centrifuged at 3000 x g for 5 minutes and the supernatants (membrane extract) were collected in microcentrifuge tubes and stored at −20 degrees Celsius. Protein concentration was quantified using the Bio-Rad protein assay (Bio-Rad).

5.5. Western Blot

Whole cell lysate or subcellular fractionation protein samples were analyzed by Western blot. 20ug of protein was added to 2x Laemelli sodium dodecyl sulfate (SDS) sample buffer, boiled at 99 degrees Celsius for 10 min, and loaded onto 4–20% Mini-PROTEAN TGX Stain-Free Protein Gels (#456–8094, Bio-Rad). Following separation, the proteins were transferred to nitrocellulose membranes (#1704156, Bio-Rad) using the Trans-Blot Turbo Transfer System (Bio-Rad), blocked for 1 hr with TBS-T containing 5% nonfat dry milk, and probed using GLUT1 (NB110–39113SS, Novus Biologicals, Littleton, CO) antibody for 12 hrs at 4 degrees Celsius, and a goat anti-rat HRP secondary antibody (#170–6515, Bio-Rad) (1:4000) for 1.5 hr at room temperature. The blot was developed by exposure to ECL reagent (#102031089, #102031090, Bio-Rad) and visualized with the ChemiDoc XRS+ System (Bio-Rad). Bands were compared to molecular weight marker protein (#161–0374, Bio-Rad). For GLUT1, intensity of bands for proteins of interest were analyzed with Image Lab Software (Bio-Rad) and normalized to total protein per Bio-Rad Gel Protocol.

5.6. Fluorometric Glucose Analog Uptake Assay

The glucose uptake and utilization of primary astrocytes cultured in normal glucose DMEM or high glucose DMEM was estimated using the Glucose Uptake Cell-Based Assay Kit (Cayman Chemical, Ann Arbor, MI, USA) according to manufacturer’s instructions. Briefly, cells were seeded at a density of 50,000 cells per well in a clear 96-well flat-bottomed dish (Corning) and incubated overnight. The next day, the cells were rinsed with PBS and incubated for 1 hr with glucose-free DMEM and fluorescent glucose analog 2-deoxy-2-[(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]-D-glucose (2- NBDG) at a concentration of 300μM. The plates were then centrifuged at room temperature for 5 min at 400g, and the supernatant was aspirated. Cell-Based Assay Buffer was added to each well (100 μL/well). The fluorescent intensity was then measured using the Cytation5 (BioTek Instruments Inc., Winooski, Vermont) (excitation/emission = 485 nm/535 nm). Fluorescent intensity was normalized to protein concentration which was measured using the Bio-Rad protein assay (Bio-Rad).

5.7. Fluorometric Glycogen Assay

NG and HG primary astrocytes were serum starved for 4 hrs in respective glucose-containing DMEM and subsequently exposed to serum-free, glucose-free DMEM. Glycogen content was measured using the Glycogen Assay Kit (ab65620, Abcam) according to manufacturer’s instructions. Fluorescent intensity was measured with the Cytation5 (Biotek) (excitation/emission = 535/587) and normalized to protein concentration measured using the Bio-Rad protein assay (Bio-Rad).

5.8. Metabolic Profiling

The Seahorse XF-96 Flux Analyzer (Seahorse Bioscience Inc. Billerica, MA) was used to determine the metabolic profile of cultured cells. The Mito Stress Test and the Glycolysis Stress Test were completed according to the manufacturer’s protocol. Briefly, the day prior to the assay astrocytes were plated on the Seahorse XF-96 microplate at a density of 25,000 cells per well. Individual cultures (n=7/group) were plated in triplicate for the assay. For the Mito Stress Test, NG and HG astrocytes were kept in their respective media. Measures of oxygen consumption rate (pmol/min) were taken before and then during the step-wise addition of oligomycin (4 μM), FCCP (1 μM), and rotenone (1 μM). For the Glycolytic Stress Test, cells were kept in glucose-free DMEM for 1 hour prior to the start of the assay. Extracellular acidification rate was then measured before and then during the step-wise addition of a saturating glucose dose (30 mM), oligomycin (4 μM) and 2-deoxyglucose (50 mM).

5.9. Statistical Analysis

Statistical analysis was performed using Graph Pad Prism. All data are expressed as mean ± standard error of mean (SEM). Student’s unpaired t-test or ANOVA was used to identify any significant differences. p < 0.05 was considered as statistically significant.

HIGHLIGHTS.

• High glucose (HG) conditioning shifts astrocytes towards glycogen storage.

• HG astrocytes break down glycogen when challenged with glucose deprivation.

• HG astrocytes do not increase GLUT1 at the membrane yet take up more glucose.

• HG astrocytes demonstrate decreased metabolic efficiency and capacity.

Abbreviations

(HG)

High Glucose

(NG)

Normal Glucose

(GLUT1)

Glucose Transporter 1

(GSK3P)

Glycogen Synthase Kinase 3 Beta

(G6P)

Glucose-6-Phosphate

(AMPK)

AMP-activated Kinase

(GS)

Glycogen Synthase

(GP)

Glycogen Phosphorylase

(PhK)

Phosphorylase Kinase

(NFI)

Normalized Fluorescent Index

(ECAR)

Extracellular Acidification Rate

(ANLS)

Astrocyte-Neuron-Lactate-Shuttle

(OCR)

Oxygen Consumption Rate