Human primary astrocytes increase basal fatty acid ... · DG) increase intracellular calcium in hindbrain astrocytes [14], which is a well-accepted marker of increased astrocyte activity

Human primary astrocytes increase basal fatty acid oxidation following

recurrent low glucose to maintain intracellular nucleotide levels

Paul G Weightman Potter1; Julia M. Vlachaki Walker1; Josephine L Robb1; John K.

Chilton1; Ritchie Williamson2; Andrew Randall3; Kate L.J. Ellacott1; Craig Beall1

1Institute of Biomedical and Clinical Sciences, University of Exeter Medical School,

RILD Building, Barrack Road, EX2 5DW, UK

2 School of Pharmacy and Medical Sciences, University of Bradford, BD7 1DP, UK

3Hatherly Laboratories, Prince of Wales Road, University of Exeter, EX4 4PS, UK

Corresponding Author:

Craig Beall

Institute of Biomedical and Clinical Sciences, University of Exeter Medical School,

RILD Building, Barrack Road, Exeter, EX2 5DW, UK

01392 408209

Email: [email protected]

.CC-BY-NC-ND 4.0 International licenseunder anot certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available

The copyright holder for this preprint (which wasthis version posted February 26, 2018. ; https://doi.org/10.1101/271981doi: bioRxiv preprint

https://doi.org/10.1101/271981

http://creativecommons.org/licenses/by-nc-nd/4.0/

ABSTRACT

Hypoglycemia is a major barrier to good glucose control in type 1 diabetes and

frequent exposure to hypoglycemia can impair awareness to subsequent bouts of

hypoglycemia. The neural changes that occur to reduce a person’s awareness of

hypoglycemia are poorly defined. Moreover, the molecular mechanisms by which

glial cells contribute to hypoglycemia sensing and glucose counterregulation require

further investigation. To test whether glia, specifically astrocytes, could detect

changes in glucose, we utilized human primary astrocytes (HPA) and U373

astrocytoma cells and exposed them to recurrent low glucose (RLG) in vitro. This

allowed measurement, with high specificity and sensitivity, of changes in cellular

metabolism following RLG. We report that the AMP-activated protein kinase

(AMPK) is activated over a pathophysiologically-relevant glucose concentration

range. We observed an increased dependency on fatty acid oxidation for basal

mitochondrial metabolism and hallmarks of mitochondrial stress including increased

proton leak and reduced coupling efficiency. Relative to glucose availability, lactate

release increased during low glucose but this was not modified by RLG, nor were

glucose uptake or glycogen levels. Taken together, these data indicate that astrocyte

mitochondria are dysfunctional following recurrent low glucose exposure, which

could have implications for hypoglycemia glucose counterregulation and/or

hypoglycemia awareness.



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INTRODUCTION

Hypoglycemia in type 1 diabetes and advanced insulin-treated type 2 diabetes remains

a significant concern for people with diabetes. For patients, it is a major barrier to

enjoying the health benefits of exercise [1, 2] and is increasingly thought to contribute

directly to an increased risk of cardiovascular events in type 1 and type 2 diabetes [3,

4]. Individuals that experience frequent hypoglycemia or have had long-standing

diabetes, produce a diminished counterregulatory response to hypoglycemia [5, 6],

often accompanied by reduced symptom awareness, termed “impaired hypoglycemia

awareness”.

Defective glucose counterregulatory responses to hypoglycemia is, at least in part,

mediated by changes in the function of critically important glucose-sensing (GS)

neurons in the ventromedial nucleus of the hypothalamus (VMH) playing an

important role; although GS neurons in the hindbrain have also been implicated [7].

In addition to neurons, the brain contains at least as many cells that lack electrically

excitable membranes. Of these the largest group is the glial cells, the largest sub-

population of which are astrocytes. As well as exhibiting a range of well-developed

cell-to-cell signaling pathways, astrocytes are increasingly recognized as important

players in central nervous system based-diseases such as obesity [8, 9], Alzheimer’s

disease [10], Parkinson’s disease [11, 12] and motor neuron disease [13].

Recent studies have demonstrated that glucoprivic stimuli such as 2-deoxyglucose (2-

DG) increase intracellular calcium in hindbrain astrocytes [14], which is a well-

accepted marker of increased astrocyte activity. In the hypothalamus, astrocyte



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activation by fasting is indicated by increased glial fibrillary acidic protein (GFAP)

expression and process complexity [15, 16], both markers of gliosis.

Astrocytes contain small amounts of glycogen that can be broken down to glucose

and released as lactate to feed active neurons. For example, astrocytic glycogen stores

can maintain axonal activity during energetic stress in white matter [17, 18] and also

contribute to memory formation [19, 20]. Brain glycogen concentrations are increased

on recovery from exercise [21], suggesting that astrocytic glycogen stores are both

physiologically and pathophysiologically regulated. Astrocytes are in intimate

apposition to the cerebral vasculature meaning that a change in glycemia may be

detected by astrocytes before neurons. Moreover, astrocytes are generally accepted as

metabolically flexible neural cells that can rapidly upregulate glycolysis to support

neuronal function, partly due to the higher expression of 6-phosphofructo-2-kinase

type 3 (PFKFB3) that can stimulate glycolysis [22]. Despite this, little is known about

the intrinsic response of human primary astrocytes to acute and recurrent low glucose

(RLG) that occurs in type 1 diabetes. Given that astrocytes are metabolically flexible

cells, we hypothesized that human astrocytes would 1) sense low glucose by

increasing the phosphorylation of the cellular energy sensor AMP-activated protein

kinase (AMPK) and 2) exhibit altered glycolytic and mitochondrial metabolism

following RLG. To do this we utilized both human primary astrocytes and U373

astrocytoma cells and measured directly components of glycolytic and mitochondrial

metabolism, lactate release, glycogen levels, intracellular nucleotide levels, and

expression of key metabolic enzymes. We demonstrate for the first time that human

astrocytes metabolically adapt by increasing basal fatty acid oxidation following a

recurrent low glucose challenge that allows intracellular nucleotide levels to be

maintained.



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RESEARCH DESIGN AND METHODS

Astrocyte isolation and cell culture.

Human primary astrocytes (HPA) were isolated from normal subventricular deep

white matter blocks immediately post-mortem following consent from next-of-kin and

with ethical approval from the North and East Devon Research Ethics Committee.

Astrocytes were isolated from tissue blocks as previously described [23], sub-cultured

and frozen stocks prepared. HPA cells were maintained in humidified incubators with

95% O2/5% CO2 in DMEM (ThermoFisher, #31885) containing 5.5 mmol/l glucose,

supplemented with 10% FBS (Gibco, 10270-106) and gentamicin (25 µg/ml;

ThermoFisher, 15750-060). U373 cells were maintained in DMEM (Sigma-Aldrich

#D5671) containing 25 mmol/l glucose, supplemented with FBS (Gibco, 10270-106),

and, penicillin and streptomycin (Gibco, 100 U/ml; 100 µg/ml). To enhance

adherence of cells during growth, flasks and petri dishes containing HPA cells were

pre-coated with poly-L-lysine (20 µg/ml; Sigma, P1274-25MG) in dH2O. 1 x 106 cells

were seeded into T75cm2 flasks and allowed to grow, and passaged after

approximately 7-10 days. For experiments, cells were seeded in to 60 mm round petri

dishes in media containing 5.5 mmol/l glucose with FBS, the day prior to study. On

the morning of study, the glucose concentration was further reduced to 2.5 mmol/l

glucose for 2 hours. Glucose concentrations were then dropped further to 0.1 mmol/l

glucose as a hypoglycemic stimulus or maintained at 2.5 mmol/l glucose. For RLG

studies over 4 days, cells were recovered in media containing high (5-5.5 mmol/l)

glucose and FBS overnight (see Fig. S1 for culture model). The reasons for this were

two fold; 1) higher glucose levels overnight (approximately 19 hours) prevented

glucose depletion from the media, which would occur if the cells were maintained at



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2.5 mM and 2) this pattern more accurately represented a model of glucose variation

seen in type 1 diabetes.

Immunoblotting

Cellular protein was isolated as previously described [24] with minor modifications.

Briefly, for acute studies 450,000 cells, or for recurrent studies 250,000 cells were

seeded on to 60 mm petri dishes and harvested at the end of experiments in 65 µl of

lysis buffer. Protein concentrations were assessed by Bradford assay. 10 µg of protein

was loaded per lane on 7-10% poly-acrylamide gels. Following electrophoresis,

proteins were transferred to nitrocellulose membranes, blocked with BSA (5% w/v) or

powered milk (5% w/v) and probed with antibodies against target proteins. Primary

antibodies used were: pThr172 AMPK (1:1,000; #2535), pSer79 acetyl-CoA

carboxylase (ACC; 1:1000; #3661), mouse α-actin (1:10,000; catalogue #A2066) was

from Sigma Aldrich. Proteins were visualized and quantified using the Odyssey

scanner (Licor, UK).

Analysis of cellular metabolism

HPA and U373 cells were seeded at 2 x 105 cells per well in Agilent Seahorse XFe96

assay plates the day prior to study. Media was exchanged for low-buffered media, pH

manually adjusted to 7.40 at 37.0°C and cells placed in an atmospheric-CO2

incubator, to remove CO2 buffering capacity, one hour prior to starting the assay.

Assay plates were loaded into the extracellular flux analyzer and studied on a 3-

minute mix, 3-minute measure cycle and compounds injected as indicated. Following

assay completion, the assay plate was removed from the analyzer, media removed and

replaced with 100 µl of 50 mmol/l NaOH to lyse cells. Protein concentrations were



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determined by Bradford assay using NaOH as a vehicle. Cellular oxygen consumption

(OCR) and extracellular acidification rates (ECAR) were normalized to total protein

concentration of each well and where appropriate, normalized to baseline values

(prior to compound injection). Mitochondrial stress tests (Agilent #103015-100) and

mitochondrial fuel flexibility tests (#103270-100) were performed as per

manufacturer’s instructions.

Measurement of intracellular nucleotides

Total ATP levels were measured using ATPlite (Perkin Elmer; #6016941) with minor

modifications as previously described [24]. ATP/ADP ratios were assayed by

luminescence assay (Sigma; #MAK135). Briefly, 1x103 cells were seeded on to black

walled 96 well plates and exposed to 2.5 mmol/l or 0.1 mmol/l glucose levels for 15-

180 minutes and nucleotide ratios assayed as per manufacturer’s instructions.

Measurement of extracellular lactate, glucose uptake and glycogen levels

Extracellular lactate was measured by assessing NADH production from NAD+ in the

presence of lactate dehydrogenase as previously described [25]. Briefly, 100 µL of

extracellular supernatant was examined against a standard curve of lactate from 0-50

nM. Glucose uptake was measured using fluorescently labeled glucose analogue 6-

(N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)amino-6-Deoxyglucose (6-NBDG). 6-NBDG

(600 µM) was added for 15 minutes in 2.5 mmol/l glucose in control conditions and

in cells exposed to RLG. Glycogen levels were measured using a fluorometric kit

from Cambridge Bioscience (Cambridge, UK) as per manufacturer’s instructions.



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Statistical analysis

For immunoblotting, densitometric values were normalized to unity to examine

relative fold change in expression. A one-sample t-test was used to determine

significant changes in phosphorylation or expression, relative to control. For

comparisons of two groups a two-tailed unpaired t-test was used and for multiple

group comparisons, a one-way ANOVA with post-hoc Bonferroni were used. To

compare the mean differences between groups split by two independent variables, a

two-way ANOVA with Bonferroni multiple comparisons tests was used. Statistical

tests performed using GraphPad Prism software (Prism 5; GraphPad Software, La

Jolla, CA, USA). Results are expressed as mean ± standard error, unless otherwise

stated. p values of <0.05 were considered statistically significant.



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RESULTS

AMP-activated protein kinase is activated by acute low glucose in human

astrocytes

AMPK activation in neurons is required for sensing hypoglycemia [26], although it is

not known whether astrocytic AMPK reacts to the same energy stress, therefore we

exposed human astrocytes to normal (2.5 mmol/l) and low brain (0.1 mmol/l) glucose

levels. In cells exposed to low glucose for 30 minutes, we noted increased

phosphorylation of AMPK at threonine-172, a site required to be phosphorylated for

full kinase activation in both the human primary astrocytes (HPA; Fig.1A,C) and

U373 cells (Fig.1B,D). This was accompanied by increased phosphorylation of acetyl

CoA carboxylase (ACC) at serine 79 (Fig.1E,F). These data are consistent with the

ubiquitous role for AMPK in detecting low glucose levels in other cells types and

now reported in glia.

RLG exposure increases mitochondrial metabolism in human astrocytes

To determine whether baseline metabolism was altered following RLG, we performed

mitochondrial stress tests on HPA and U373 cells following RLG (see Fig. S1 for

RLG model). This was performed at euglycemic glucose levels post RLG to observe

any cellular adaptations. Firstly, we observed significantly higher baseline oxygen

consumption rate (OCR) in both HPA (Fig. 2A,C) and U373 cells (Fig. 2B,D) exposed

to RLG (3 versus 0 prior bouts of low glucose), indicating persistent adaptations in

cellular metabolism. Oligomycin was then added to block ATP synthase, FCCP to

stimulate maximal respiration, and rotenone and antimycin A were added to inhibit

complex I and III, respectively. This allowed calculation of coupling efficiency,

which was significantly decreased in HPA cells only (Fig. 2E,F). However, both cell



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types displayed increased proton leak (Fig. 2G,H), indicating mitochondrial

dysfunction. Despite the functional evidence of mitochondrial dysfunction, we saw no

changes in the morphology of the filamentous mitochondrial networks between the

treatment groups as examined using MitoTrackerRed (Fig. S2A,B).

In addition to changes in OCR, we also noted significantly elevated basal extracellular

acidification rate (ECAR) in HPA cells exposed to RLG (Fig. 2I,K), indicating

enhanced glycolysis following RLG. This did not occur within U373 cells following

RLG (Fig. 2J,L).

Astrocytes exposed to RLG display enhanced glycolysis on recovery of glucose

level

Given the increased basal ECAR in HPA cells exposed to RLG, we next examined

glycolytic re-activation after an acute glucose withdrawal (~80 minutes) in HPA cells

following antecedent RLG. Under control conditions, we noted that the ECAR

(glycolysis) response to glucose was concentration dependent (Fig. 3A-C), although

ECAR was not significantly higher at 5.5 mmol/l glucose than 2.5 mmol/l glucose.

Interestingly, the ECAR response to glucose in cells exposed to RLG (3 bouts of low

glucose) was significantly greater than control (0 bouts of low glucose), indicating an

enhanced glycolytic capacity (Fig. 3A-C). This only occurred at euglycemic and

hyperglycemic levels. Consistent with enhanced glycolytic activity, we also noted a

significant reduction in oxygen consumption rate (OCR) following glucose injection,

indicative of a shift away from mitochondrial metabolism towards glycolysis (Fig.3D-

F). Correspondingly, the reduction in OCR following glucose recovery was

augmented in HPA cells exposed to RLG. Taken together these data suggest an



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enhanced Warburg effect in HPA cells following recovery of glucose levels. In U373

cells, RLG did not alter ECAR between groups (control vs RLG) on glucose recovery.

However, the re-activation of ECAR on delivery of glucose was not concentration-

dependent and in U373 cells the relative increase in ECAR was twice as large as that

in HPA cells (Fig. S3). These studies show, obtaining primary cells from source is

preferably to transformed cells.

Fatty acid oxidation (FAO) is increased in human astrocytes following RLG

To assess potential changes to mitochondrial fuel usage following RLG we performed

mitochondrial fuel flexibility tests; achieved by sequential selective inhibition of the

mitochondrial pyruvate carrier (MPC; glucose oxidation pathway), glutaminase

(glutamine oxidation pathway) and carnitine palmitoyltransferase 1 (CPT1; fatty acid

oxidation pathway). Surprisingly, there was no change in mitochondrial dependency,

capacity or flexibility to metabolize pyruvate (Fig. 4A-C). This suggests that in

astrocytes alterations to glucose metabolism are upstream of the transport of pyruvate

into the mitochondria in astrocytes. There was a non-significant trend towards

reduced glutamine dependency at baseline following RLG (Fig. 4D), with no

significant change to the glutamine oxidation capacity (Fig. 4E). Combined, this

produced a significant increase in glutamine flexibility (Fig. 4F). Interestingly, RLG

significantly increased metabolic dependency on fatty acids (FA) but with no change

in FAO maximum capacity, thereby causing a decrease in flexibility (Fig. 4G-I).

These data demonstrate that RLG induces mitochondrial adaptations to fuel sources

consistent with a shift towards increased reliance on FAO and an enhanced flexibility

to utilize glutamine.



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Human astrocytes maintain energy production (total ATP and ATP/ADP ratio)

during low glucose conditions

To examine the consequences of acute low glucose and RLG for astrocyte energy

content, we measured total ATP and the ATP/ADP ratio. These measurements were

made both in the presence of acute low glucose and following RLG. Interestingly, we

found that total ATP levels did not significantly change in HPA (Fig. 5A) or U373

cells (Fig. 5B) during acute low glucose exposure (0.1 mmol/l) lasting 3 hours.

Despite the increased basal mitochondrial and glycolytic metabolism described above,

total ATP levels following RLG were also comparable to control (Fig. 5A,B).

Similarly, the ATP/ADP ratio was not significantly different following 3 hours of 0.1

mmol/l glucose, nor was the basal ratio altered following RLG (Fig. 5C,D). These

data indicate that the total ATP content and ATP/ADP ratio are well defended in

astrocytes, at least in response to up to 3 hours of low glucose, and that changes to

glycolytic and mitochondrial metabolism may be compensatory in order to sustain

intracellular nucleotide levels.

RLG does not alter glucose uptake or glycogen levels in human astrocytes

Western blotting demonstrated that the levels of key glycolytic markers GLUT1,

hexokinase I (HKI), HKII, HKIII and phosphofructokinase (PFK) were not altered by

acute or recurrent low glucose in human astrocytes (Fig. S4).

Lactate release from HPA and U373 cells decreased by 25-50% during 3 hours of low

glucose exposure (Fig. 6A,B), although this was a substantial decrease, this was

proportionally much less than the 25-fold reduction in glucose availability. Hence,

when corrected for glucose availability, there was a large relative increase in lactate



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release (Fig. 6C,D); however, this was not different between control and RLG-treated

cells.

We next assessed the degree of glucose uptake by human astrocytes using the

fluorescently labelled glucose analogue, 6-(N-(7-Nitrobenz-2-oxa-1,3-diazol-4-

yl)amino-6-Deoxyglucose (6-NBDG). HPA cells exposed to RLG showed no change

in 6-NBDG uptake (Fig. 6E), indicating that the persistent changes in mitochondrial

oxygen consumption were not simply mediated by increased glucose uptake.

Correspondingly, we did not observe any changes in astrocytic glycogen content

following RLG (Fig. 6F).

Correlating with our mitochondrial imaging studies, the levels of mitochondrial

markers succinate dehydrogenase (SDH) and the voltage-dependent anion channel

(VDAC) were not altered by RLG, suggesting no change in total mitochondrial

content (Fig. S5). Fatty acid synthase (FAS) and carnitine palmitoyltransferase 1a

(CPT-1a) expression were also not altered, indicating that changes in FA-dependency

following RLG are likely to be mediated by metabolic flux rather than expression of

enzymes within the FAO pathway.



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DISCUSSION

Our studies shed new light on the previously uncharacterized intrinsic metabolic

changes in human glial cells in response to acute and recurrent low glucose. We

demonstrate that astrocytes, in the absence of physical and chemical signals from

neurons, react to low glucose by increasing the activation of the key metabolic sensor

AMPK. Changes in AMPK pathway phosphorylation occur within a

pathophysiologically-relevant glucose concentration range, similar to that which

affects CNS glucose-sensing neurons [28, 29].

It is well-accepted that astrocytes are more metabolically flexible than neurons, partly

due to the higher expression of PFKFB3, which can accelerate glycolysis during

stress [22]. Therefore, to examine specifically the changes in astrocyte metabolism

following recurrent low glucose, we measured markers of mitochondrial and

glycolytic metabolism in human primary astrocyte mono-cultures. Importantly, we

found that daily prior bouts of low glucose (4 x 0.1 mmol/l glucose) lasting 3 hours,

were sufficient to increase basal mitochondrial and glycolytic metabolism and sustain

intracellular ATP levels. This increased basal mitochondrial metabolism is likely

mediated, at least in part, by an increased contribution of FAO to basal oxygen

consumption. Correlating with this hypothesis, we noted increased mitochondrial

proton leak and reduced mitochondrial coupling efficiency in astrocytes exposed to

RLG. Importantly, FAO is known to increase uncoupling, as FAO generates increased

levels of reactive oxygen species (ROS)/superoxide [31] that can increase proton leak

via activation of uncoupling proteins or the ATP/ADP antiporter (ANT; [32]).

Previous work has demonstrated that in vivo, genes for β-oxidation are induced by

acute hypoglycemia but this fails following recurrent hypoglycemia (RH)[33]. Our



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data, in astrocyte monocultures contrasts with these measurements taken from whole

rodent mediobasal hypothalamus, which include both neurons and glia. Therefore, it

is possible that cell type-specific changes are masked when measuring mRNA

expression from whole tissue sections. Despite the evidence of mitochondrial stress

and in contrast to our expectation, examination of the filamentous mitochondrial

networks revealed no change in either mitochondrial size or number following acute

low glucose or following RLG. Indeed, mitochondria exposed to RLG generally

displayed a normal filamentous network, with little or no evidence of mitochondrial

fragmentation. These data suggest that astrocyte mitochondria successfully adapt to

maintain energy production in response to repeated bouts of low glucose availability.

Previous studies in rats found no change in brain glycogen levels following RH [34]

or any correlation between brain glycogen content and impaired awareness of

hypoglycemia in humans [35]. However, other studies suggest brain glycogen content

is increased following recovery from insulin-induced hypoglycemia [36] and

following recurrent 2-DG induced glucoprivation in mice [37]. Our data clearly

demonstrate that astrocytes intrinsically adapt by increasing cellular metabolism in

response to recurrent energy stress. Specifically, rates of extracellular acidification (a

marker for glycolysis) following glucose “reperfusion” were elevated in cells exposed

to RLG, suggesting a augmented re-activation of glycolysis. However, despite

glycolysis being a major determinant of glycogen synthesis, we found no significant

change in astrocytic glycogen levels following RLG. In our model, we measured

extracellular lactate accumulation (which can be generated from glycogen breakdown

during aglycemia [18]) in conditioned media from astrocytes over a 3 hour period.

After correcting for glucose availability, lactate release increased substantially during



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low glucose exposure, although the magnitude of this response was not significantly

altered following RLG. We cannot rule out, however, the possibility that more subtle

changes in lactate and glycogen occur during the transition to low glucose or on

glucose recovery. Moreover, before definitively ruling out a role for astrocytic

glycogen and subsequent lactate release, a more replete in vitro model, containing

both astrocytes and neurons should be tested, thus facilitating the inclusion of

neurotransmitter dynamics into the model. The ability to produce increasingly

credible human neurons from iPSC sources could potentially enable such

investigations to be performed in an in vitro human system.

Astrocytes play a key role in maintaining glutamatergic neurotransmission through

the glutamate-glutamine cycle (for recent review see [38]). Importantly, astrocytic

glutamate clearance requires co-transport of three Na+ ions [39], meaning that, via the

ATP-dependent Na+/K+ ATPase, there is a significant metabolic cost for astrocytes to

maintain their Na+ homeostasis, estimated to be approximately 20% of astrocyte ATP

production [40]. This process may become particularly costly during hypoglycemia.

Moreover, glutamate stimulation of astrocytes produces mitochondrial dysfunction,

decreasing mitochondrial spare respiratory capacity and increasing lactate production

[41]. Furthermore, recent observations suggest that astrocytic glutamate recycling is

diminished following RH, a change most likely mediated by reduced glutamate

uptake [42]. Our data add to these findings and suggest that metabolic changes within

astrocytes, could play a part in defective counterregulatory responses following

recurrent hypoglycemia. In support of this, astrocytes are increasingly recognized as

playing an important role in regulation of whole body metabolism. For example,

chemogenetic activation (by increasing intracellular Ca2+) of astrocytes in the arcuate



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nucleus increased feeding in mice and conversely, sequestering astrocytic Ca2+,

decreased food intake [15]. Moreover, modulation of inflammatory signaling in

astrocytes also modulates feeding in response to a high fat diet [43, 44], further

supporting a role for astrocytes in regulating energy sensing neurons of the

hypothalamus. Within the hindbrain, astrocytes can detect hypoglycemia to regulate

adjacent neurons to increase gastric emptying in response to a glucoprivic stimulus

[14]. Astrocytes have also been shown to decrease hyperglycemia in a rodent model

of type 1 diabetes [45] suggesting that astrocytes may play a fundamental role in

regulating whole body glucose levels. In conclusion, our data further support the

evidence that glial cells are likely to play a role in detecting hypoglycemia and that

specifically changes in astrocyte mitochondrial function (increased fatty acid

oxidation) could play a role in defective glucose counterregulation. This study also

highlights the need for more investigation into neuron-glial interactions during and

following recurrent hypoglycemia.



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Acknowledgements

This study was funded by grants from Diabetes UK (RD Lawrence Fellowship to CB;

13/0004647), the Medical Research Council (MR/N012763/1) to KLJE, ADR and

CB, a Mary Kinross Charitable Trust PhD studentship to CB, ADR and RW to

support PWP. Additional support for this work came from awards from the British

Society for Neuroendocrinology (to CB and KLJE), Society for Endocrinology (CB),

Tenovus Scotland (CB) and the University of Exeter Medical School (CB and KLJE).

AR was also supported by a Royal Society Industry Fellowship. We also wish to

thank Nicholas Gutowski and Janet Holley for kindly gifting the human primary

astrocytes. We also wish to thank Nick Howe for guidance with Extracellular Flux

Analysis. Parts of this study were presented at the American Diabetes Association

77th Scientific Sessions and at the Diabetes UK Annual Professional Conference

2017.

Author Contributions

P.G.W.P, J.M.V.W., J.L.R., R.W., A.D.R. and C.B. researched data. J.K.C., A.D.R.,

P.G.W.P, K.L.J.E., and C.B. contributed to experimental design, analysis and writing

of the manuscript. C.B. conceived the study and is the guarantor of this work and, as

such, had full access to all the data in the study and takes responsibility for the

integrity of the data and the accuracy of the data analysis.

Conflict of Interest Statement

The authors have no conflict of interest to declare



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Figure Legends

Figure 1. Acute low glucose increases AMP-activated protein kinase (AMPK)

pathway activation. Representative immunoblots from human primary astrocyte

(HPA, A; n=8) and U373 astrocytoma cells (B; n =3) exposed to 2.5 or 0.1 mmol/l

glucose media for 30 minutes. C, D. Densitometric analysis of immunostaining for

pT172 AMPK normalized to actin as a loading control. E, F. Densitometric analysis

of immunostaining for pS79 ACC normalized to actin as a loading control.

*P<0.05;***P<0.001. One-sample t-test in comparison to control.

Figure 2. Recurrent low glucose (RLG) increases baseline mitochondrial

metabolism and reduces mitochondrial coupling efficiency. Oxygen consumption

rate (OCR) of human primary astrocytes (HPA; A; 36-39 across three separate

assays) and U373 cells (B) following RLG (n=40-42, across across separate assays).

Cells were exposed to oligomycin (10 µM), FCCP (5 µM) and a combination of

rotenone and antimycin A (5 µM). C, D. Mean basal OCR in control and RLG treated

astrocytes. E, F. Coupling efficiency calculated from the ratio of oligomycin sensitive

OCR and basal OCR expressed as a percentage. G, H. Proton leak, calculated from

the oligomycin-insensitive (i.e. not ATP-synthase linked) OCR minus non-

mitochondrial respiration, from HPA cells and U373 cells. I, J. Extracellular

acidification rate (ECAR) analysis measured during mitochondrial stress tests. K, L.

Mean baseline ECAR in HPA and U373 cells following RLG, respectively. *P<0.05;

***P<0.001. Two-tailed students t-test.

Figure 3. Recurrent low glucose (RLG) increases glycolytic re-activation after

acute low glucose exposure. Extracellular acidification rate (ECAR) increased in



https://doi.org/10.1101/271981


human primary astrocyte (HPA) cells on re-introduction of 0.5 mmol/l glucose (A),

2.5 mmol/l glucose (B) and 5.5 mmol/l glucose (C). Oxygen consumption rate (OCR)

decreased on addition of 0.5 mmol/l glucose (D), 2.5 mmol/l glucose (E) and 5.5

mmol/l glucose (F). *P<0.05; **P<0.01; ***P<0.001 (n=8-10).

Figure 4. Recurrent low glucose (RLG) alters human primary astrocyte (HPA)

mitochondrial fuel flexibility to increase dependency on fatty acids for basal

oxidation. A-C. Inhibition of mitochondrial pyruvate carrier using UK5099 (2 µM) to

measure dependency, maximum capacity and flexibility within the glucose oxidation

pathway. D-F. Inhibition of the glutaminase using BPTES (3 µM) to measure changes

in the glutamine oxidation pathway following RLG. G-I. Inhibition of carnitine

palmitoyltransferase 1 CPT1 using etomoxir (4 µM) to measure changes in the fatty

acid (FA) oxidation pathway following RLG. *P<0.05 (n=21-32). Dependency:

contribution of that pathway to baseline oxygen consumption rate. Capacity:

maximum ability to oxidize through that pathway when two other oxidative pathways

are inhibited. Flexibility: calculated from the difference between dependency and

capacity.

Figure 5. Acute and recurrent low glucose (RLG) does not modify intracellular

ATP and ATP/ADP ratios. Total intracellular ATP levels in human primary

astrocytes (HPA; A, n=6) and U373 astrocytoma cells (B; n=6) exposed to different

glucose concentrations for 3 hours. Intracellular ATP/ADP ratios in HPA (C; n=6)

and U373 astrocytoma cells (D; n=4) exposed to 2.5 or 0.1 mmol/l glucose for 3

hours.



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Figure 6. Effect of recurrent low glucose (RLG) on human astrocyte lactate

release, glucose uptake and glycogen levels. Extracellular lactate levels measured

from conditioned media from human primary astrocytes (HPA; A; n=8) and U373 (B;

n=6) following exposure to 2.5 or 0.1 mmol/l glucose containing media for 3 hours.

Extracellular lactate levels normalized to glucose availability from conditioned media

from HPA (C; n=8) and U373 (D; n=6) following exposure to 2.5 or 0.1 mmol/l

glucose containing media for 3 hours. E. Fluorescent signal from labeled glucose

analogue 6-NBDG incubated with HPA cells for 15 minutes. F. Glycogen content of

HPA cells in control and following exposure to RLG (n=5). *P<0.05; **P<0.01;

***P<0.001.

Supplementary Figure 1. Illustration of the recurrent low glucose model. Over 4

days, cells are exposed to 0, 1, 3 or 4 bouts of 0.1 mM glucose, representing control,

acute low glucose, antecedent low glucose or recurrent low glucose, respectively.

Supplementary Figure 2. Acute and recurrent low glucose does not alter

mitochondrial number or length. A. Representative confocal images of human

primary astrocytes (HPA) cells exposed to 2.5 or 0.1 mM glucose for 3 hours

following control or recurrent low glucose exposure (RLG). Raw confocal images are

shown on the left hand side, with extracted signal images shown on the right. B.

Quantification of median object size (in pixels) using a custom MatLab script (15

images across 3 separate experiments).

Supplementary Figure 3. Recurrent low glucose (RLG) does not alter glycolytic

re-activation after acute low glucose exposure in U373 cells. Extracellular



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acidification rates (ECAR) increased in U373 cells on re-introduction of 0.5 mmol/l

glucose (A), 2.5 mmol/l glucose (B) and 5.5 mmol/l glucose (C) although this was not

significantly different between control and recurrent low glucose (RLG) treated cells.

Oxygen consumption rate (OCR) decreased on addition of 0.5 mmol/l glucose (D),

2.5 mmol/l glucose (E) and 5.5 mmol/l glucose (F), which was not significantly

different between control and RLG (n=8-10).

Supplementary Figure 4. Expression of key mitochondrial and glycolytic

markers are not altered by acute or recurrent low glucose exposure (RLG).

Glucose transporter 1 (GLUT-1), hexokinase I (HKI), hexokinase II (HKII),

hexokinase III (HKIII), phosphofructokinase [platelet; PFK(P)], voltage dependent

anion channel (VDAC), succinate dehydrogenase A (SDHA), carnitine

palmitoyltransferase 1α (CPT1α) and fatty acid synthesis (FAS) were not altered by

acute or recurrent low glucose exposure (n=3-4).



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Figure 1

CpT172 AMPK/Actin

[Glucose] (mM)2.5 0.1

*

pS79 ACC

pT172 AMPK

Actin

2.5 0.1

[Glucose] (mM)

A

pS79 ACC

pT172 AMPK

Actin

2.5 0.1

[Glucose] (mM)

B

pT172 AMPK/Actin

2.5 0.10

2

4

6

*

Fo

ld C

han

ge

inP

ho

sph

ory

lati

on

(A

U)

[Glucose] (mM)

Fo

ld C

han

ge

inP

ho

sph

ory

lati

on

(A

U)

2.5 0.10

2

4

6

HPA U373

EpS79 ACC/Actin

[Glucose] (mM)2.5 0.1

***

[Glucose] (mM)

Fo

ld C

han

ge

inP

ho

sph

ory

lati

on

(A

U)

0

1

2

3

4

5

pS79 ACC/Actin

2.5 0.10

1

2

3

4

5

*

Fo

ld C

han

ge

in

Ph

osp

ho

ryla

tio

n (

AU

)

D

F



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R/AOligo FCCP

B

0 20 40 60 80 1000

2

4

6

U373

Time (minutes)

OC

R (

pm

ol/m

in/μ

g) Control

RLGO

CR

(p

mo

l/min

/μg

)

Control RLG0.0

0.5

1.0

1.5

2.0

2.5

*

HPA Basal Mitochondrial Respiration

C

***

Per

cen

tag

e (

%)

HPA Coupling efficiency

Control RLG0

20

40

60

80

***

E U373 Coupling efficiencyP

erce

nta

ge

(%

)

Control RLG0

20

40

60

80

F

K

Control RLG

Basal ECAR

EC

AR

(m

pH

/min

/μg

)

0

1

2

3

4

5L

Control RLG

HG HPA Proton Leak

OC

R (

pm

ol/m

in/μ

g)

Control RLG0.0

0.2

0.4

0.6

0.8

1.0

***

U373 Proton Leak

OC

R (

pm

ol/m

in/μ

g)

Control RLG0.0

0.2

0.4

0.6

0.8

1.0

**

Figure 2

U373 Basal Mitochondrial Respiration

OC

R (

pm

ol/m

in/μ

g)

Control RLG0.0

0.5

1.0

1.5

2.0

2.5

D

AR/AOligo FCCP

0 20 40 60 80 1000

2

4

6

HPA

Time (minutes)

OC

R (

pm

ol/m

in/μ

g) Control

RLG

J

0 20 40 60 80 1000

2

4

6

Extracellular acidification rate

Time (minutes)

EC

AR

(m

pH

/min

/μg

)

RLGControl

I

0 20 40 60 80 1000

2

4

6

Extracellular acidification rate

Time (minutes)

EC

AR

(m

pH

/min

/μg

)

RLGControl



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Figure 3

A B C

D E F

Time (min)

ΔE

CA

R (

mp

H/m

in/μ

g)

0 20 40 60 800

10

20

30

40

ControlRLG

0.5 mM glucose

0.5 mM Glu

2.5 mM glucose

Time (min)

ΔE

CA

R (

mp

H/m

in/μ

g)

0 20 40 60 800

10

20

30

40

* *** ** **

** ** **

2.5 mM Glu

Time (min)

ΔE

CA

R (

mp

H/m

in/μ

g)

0 20 40 60 800

10

20

30

40

* * * * * * *

5.5 mM glucose

5.5 mM Glu

ControlRLG

0.5 mM glucose

Time (min)

ΔO

CR

(p

mo

l/min

/μg

) 20 40 60 80

-30

-20

-10

0

0.5 mM Glu

2.5 mM glucose

Time (min)

ΔO

CR

(p

mo

l/min

/μg

) 20 40 60 80

-30

-20

-10

0

* ** ** ***************2.5 mM Glu

5.5 mM glucose

Time (min)

ΔO

CR

(p

mo

l/min

/μg

) 20 40 60 80

-30

-20

-10

0

* * *5.5 mM Glu



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Figure 4

GLN Capacity

% C

apac

ity

Control RLG0

20

40

60E GLN Flexibility

% F

lexi

bili

ty

Control RLG0

10

20

30

40*

F

FA Dependency

% D

epen

den

cy

Control RLG0

5

10

15

20

25G

*FA Capacity

% C

apac

ity

Control RLG0

20

40

60

80H FA Flexibility

% F

lexi

bili

ty

Control RLG0

20

40

60I

*

GLC Dependency

% D

epen

den

cy

Control RLG0

10

20

30

40A GLC Capacity

% C

apac

ity

Control RLG0

20

40

60

80

100B GLC Flexibility

% F

lexi

bili

ty

Control RLG0

20

40

60C

GLN Dependency

% D

epen

den

cy

Control RLG0

10

20

30 p=0.09D



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Figure 5

A HPA Total ATP

No

rmal

ised

Lu

min

esce

nce

(A

U)

2.5 0.1 2.5 0.10

50

100

150

Control RLG

[Glu] (mM)

B U373 Total ATP

No

rmal

ised

Lu

min

esce

nce

(A

U)

2.5 0.1 2.5 0.10

50

100

150

Control RLG

[Glu] (mM)

DU373 ATP/ADP Ratio

AT

P/A

DP

rat

io

2.5 0.1 2.5 0.10

2

4

6

Control RLG

[Glu] (mM)

CHPA ATP/ADP Ratio

AT

P/A

DP

rat

io

2.5 0.1 2.5 0.10

2

4

6

Control RLG[Glu] (mM)



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F HPA Glycogen Content

[Gly

cog

en]

(um

ol/m

g)

Control RLG0

5

10

15

HPA 6-NBDG Uptake

Flu

ore

scen

ce (

AU

)

Control RLG0

20000

40000

60000

E

Figure 6

DU373 eLactate relativeto glucose availability

Lac

tate

(n

mo

l/mM

)

0

2000

4000

6000

******

Control2.5 0.1

RLG2.5 0.1

CHPA eLactate relativeto glucose availability

Lac

tate

(n

mo

l/mM

)

0

2000

4000

6000***

***

Control2.5 0.1

RLG2.5 0.1

B U373

Lac

tate

(n

mo

l/mg

)

0

200

400

600

800

1000

* **

Control2.5 0.1

RLG2.5 0.1[Glu] (mM) [Glu] (mM)

[Glu] (mM)[Glu] (mM)

A HPA

0

200

400

600

800

1000

Control2.5 0.1

RLG2.5 0.1

Lac

tate

(n

mo

l/mg

) .CC-BY-NC-ND 4.0 International licenseunder anot certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available


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Human primary astrocytes increase basal fatty acid ... · DG) increase intracellular calcium in hindbrain astrocytes [14], which is a well-accepted marker of increased astrocyte activity