Regulation of glycolysis by the hypoxia‐inducible factor (HIF): implications for cellular physiology

Abstract

Under conditions of hypoxia, most eukaryotic cells can shift their primary metabolic strategy from predominantly mitochondrial respiration towards increased glycolysis to maintain ATP levels. This hypoxia‐induced reprogramming of metabolism is key to satisfying cellular energetic requirements during acute hypoxic stress. At a transcriptional level, this metabolic switch can be regulated by several pathways including the hypoxia inducible factor‐1α (HIF‐1α) which induces an increased expression of glycolytic enzymes. While this increase in glycolytic flux is beneficial for maintaining bioenergetic homeostasis during hypoxia, the pathways mediating this increase can also be exploited by cancer cells to promote tumour survival and growth, an area which has been extensively studied. It has recently become appreciated that increased glycolytic metabolism in hypoxia may also have profound effects on cellular physiology in hypoxic immune and endothelial cells. Therefore, understanding the mechanisms central to mediating this reprogramming are of importance from both physiological and pathophysiological standpoints. In this review, we highlight the role of HIF‐1α in the regulation of hypoxic glycolysis and its implications for physiological processes such as angiogenesis and immune cell effector function. image

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J Physiol 00.0 (2020) pp 1–15 1
The Journal of Physiology
TOPICAL REVIEW
Regulation of glycolysis by the hypoxia-inducible factor
(HIF): implications for cellular physiology
S. J. Kierans1,2 and C. T. Taylor1,2
1Conway Institute of Biomolecular and Biomedical Research, University College Dublin, Belfield, Dublin 4, Ireland
2School of Medicine, University College Dublin, Belfield, Dublin 4, Ireland
Edited by: Ian Forsythe & Paul Greenhaff
Cormac Taylor is Professor of Cellular Physiology at the School of Medicine and the Conway Institute,
University College Dublin. Previous appointments include a postdoctoral fellowship at Brigham and
Wome n ’s Ho s p i t a l a n d Har va rd Me d i c a l S choo l (Bost o n ) . P rof e s s o r Ta y lor was t h e r e c i p ient o f the Na t u r e
midcareer mentorship award (2014). The Taylor lab investigates the impact of hypoxia and hypercapnia
on cellular pathways and function in the context of inflammation and immunity. Sarah Kierans is a PhD
student working in the Taylor laboratory on the pathways regulating glycolysis in hypoxia in tumour cells
and immune cells.
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2 S. J. Kierans and C. T. Taylor J Physiol 00.0
Abstract Under conditions of hypoxia, most eukaryotic cells can shift their primary metabolic
strategy from predominantly mitochondrial respiration towards increased glycolysis to maintain
ATP levels. This hypoxia-induced reprogramming of metabolism is key to satisfying cellular
energetic requirements during acute hypoxic stress. At a transcriptional level, this metabolic
switch can be regulated by several pathways including the hypoxia inducible factor-1α(HIF-1α)
which induces an increased expression of glycolytic enzymes. While this increase in glycolytic flux
is beneficial for maintaining bioenergetic homeostasis during hypoxia, the pathways mediating
this increase can also be exploited by cancer cells to promote tumour survival and growth,
an area which has been extensively studied. It has recently become appreciated that increased
glycolytic metabolism in hypoxia may also have profound effects on cellular physiology in hypoxic
immune and endothelial cells. Therefore, understanding the mechanisms central to mediating this
reprogramming are of importance from both physiological and pathophysiological standpoints.
In this review, we highlight the role of HIF-1αin the regulation of hypoxic glycolysis and its
implications for physiological processes such as angiogenesis and immune cell effector function.
(Received 6 August 2020; accepted after revision 25 September 2020; first published online 2 October 2020)
Corresponding author C. T. Taylor: Conway Institute of Biomolecular and Biomedical Research, University College
Dublin, Belfield, Dublin D4, Ireland. Email: cormac.taylor@ucd.ie
Abstract figurelegend Schematic outlining the functional physiological consequences of enhanced glycolytic metabolism
in endothelial cells and immune cells.
Introduction
Glycolysis, which takes place in the cytoplasm, describes
the anaerobic conversion of a single molecule of glucose
into two molecules of pyruvate. The metabolism of glucose
is achieved through the oxygen-independent activities
of 10 metabolic enzymes which comprise the glycolytic
pathway to result in the generation of biochemical energy
equivalents in the form of adenosine triphosphate (ATP),
the concurrent reduction of nicotinamide adenosine
dinucleotide (NAD) to NADH and the generation of
pyruvate (Fig. 1). In the presence of sufficient levels
of molecular oxygen (O2), the pyruvate generated is
further metabolised within the mitochondria by the
tricarboxylic acid (TCA) cycle which provides electron
carriers to the electron transport chain (ETC) to generate
afurther36moleculesofATPpermoleculeofglucose
entering the glycolytic pathway. In the steady state, the
combined activities of glycolysis, the TCA cycle and
the ETC usually results in the generation of sufficient
levels of ATP to meet the bioenergetic requirements
of the cell, tissue and organism and thereby maintain
homeostasis.
The glycolytic pathway is evolutionarily conserved
across all kingdoms, including archaeal (Selig et al. 1997),
fungal (Moses, 1959), protozoal (Becker & Geiman, 1955;
Bragg & Reeves, 1962), bacterial (Kornberg, 1976) and
metazoan species (Wan et al. 2015), in addition to being
well described in plant metabolism (Plaxton, 1996). The
rate of glycolysis is reliant on the availability and sub-
sequent uptake of glucose. In mammalian cells, glucose
uptake is achieved by two means: the active transport
of glucose as evident in the mammalian small intestinal
epithelial cells via sodium-dependent glucose trans-
porters (SGLT), or alternatively, via facultative diffusion
through glucose transporters (GLUT) located within
the cell membrane of multiple cell types (Pessin &
Bell, 1992). Once taken up into the cell, glucose is
oxidised in a stepwise manner, at a rate defined by
the rate-limiting enzymes of glycolysis. These enzymes;
hexokinase, phosphofructokinase and pyruvate kinase,
are allosterically regulated and are understood to have
important roles in the regulation of glycolytic flux (Jurica
et al.1998;Kemp&Gunasekera,2002;Chanetonet al.
2012).
Glycolysis in hypoxia
Under conditions where oxygen demand by a tissue
exceeds its supply (hypoxia), cells reduce their
reliance upon O2-dependent mitochondrial oxidative
phosphorylation (OXPHOS) and preferentially use
the O2-independent glycolytic pathway to maintain
sufficient ATP production in order satisfy bio-energetic
requirements. This metabolic switch in response to hypo-
xia is initiated by the allosteric control of glycolytic
enzymes by ATP. Under hypoxic conditions, whereby
OXPHOS has become inhibited, a low level of ATP
is produced which effectively decreases the ATP/AMP
(adenosine monophosphate) ratio. This reduction in
energy charge reduces the allosteric inhibition of
ATP on glycolytic enzyme phosphofructokinase (PFK)
(Henderson, 1969). Activated PFK utilises ATP to
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J Physiol 00.0 HIF-dependent regulation of glycolysis 3
produce fructose-1,6-bisphosphate and promote flux
through the glycolytic pathway (Fig. 1). ATP further
allosterically inhibits pyruvate kinase to reduce the rate
of glycolysis when energy charge is high. Therefore,
under conditions of hypoxia, the reduction in energy
charge reduces the allosteric inhibition of ATP on
pyruvate kinase to result in the generation of pyruvate
from phosphoenolpyruvate (Fig. 1). Pyruvate kinase is
also activated by fructose-1,6-bisphosphate (feed-forward
regulation) to promote complete flux through the
glycolytic pathway.
The allosteric regulation of glycolysis under hypo-
xic conditions is subsequently followed by the trans-
criptional upregulation of glucose transporters and
glycolytic enzymes by the hypoxia inducible factor
(HIF) transcription factor. This upregulation of glycolytic
enzymes allows for an increased flux though the pathway,
and is thus advantageous in maintaining cellular ATP
production under hypoxic conditions. When oxygen
balance (and ATP/AMP ratio) is restored, cells can usually
revert their primary metabolic strategy to OXPHOS, a
phenomenon known as the Pasteur effect (Pasteur, 1861).
Figure 1. Schematic overview of the glycolytic pathway
Glucose is taken up into the cell from the extracellular space via glucose transporters (GLUT) located within the
cellular membrane. Glucose is oxidised in a stepwise manner via the actions of glycolytic enzymes (orange text).
The glycolytic pathway relies on the investment of 2 molecules of ATP to oxidize glucose (preparatory phase, A)
and allows for the concurrent generation of 2 molecules of NADH, 2 H+,2moleculesofH
2O, 2 molecules of
pyruvate and a net gain of 2 ATP per unit of glucose (pay-off phase; B).
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4 S. J. Kierans and C. T. Taylor J Physiol 00.0
While this hypoxia-induced increase in glycolytic flux
is beneficial in promoting cell survival during a hypoxic
insult, and is thus adaptive in nature, the mechanisms
surrounding this increased glycolytic flux can also be
manipulated by cancer cells to promote their survival
even in the presence of sufficient levels of O2(known
as aerobic glycolysis or the Warburg effect; Warburg,
1956; Fig. 2). Therefore, the mechanisms central to
mediating this metabolic switch are of key importance
in both health and disease. To date multiple mechanisms
have been described as important regulators of glycolysis
under hypoxic conditions, including those dependent
on the actions of the HIF pathway (Seagroves et al.
2001), the PI3K/Akt pathway (Xie et al. 2019) and
hypoxia-dependent post-translational modification of
glycolytic enzymes (Agbor et al.2011),amongstothers.
In this review, we will focus on the role of HIF in the
physiological regulation of glycolysis in hypoxic cells.
The HIF pathway. HIF is a heterodimeric protein
consisting of an oxygen sensitive αsubunit (HIF-1α,
HIF-2α,HIF-3α)andaconstitutivelyexpressedβsubunit
[HIF-1β/Aryl hydrocarbon receptor nuclear translocator
(ARNT)]. Of the three HIFα-homologues identified in
higher metazoans, HIF-1αis responsible for regulating
glycolysis in response to hypoxia (Hu et al.2003).
In the presence of sufficient levels of O2(normoxia),
HIF-1αhas a short half-life, estimated at about 5–10 min
(Wang et al.1995).Innormoxicconditions,theHIF-1α
protein becomes rapidly degraded as a result of the
oxygen-dependent hydroxylation at conserved proline
residues (Pro402 and Pro564 )oftheαsubunit (Ivan
et al.2001;Jaakkolaet al.2001;Yuet al.2001).This
hydroxylation is carried out by members of the prolyl
hydroxylase domain (PHD) family, of which three major
isoforms have been characterised in mammalian cells.
Hydroxylation of HIF-1αpromotes its binding to the
von Hippel-Lindau (VHL) protein, a component of the
E3 ubiquitin ligase complex, which leads to the poly-
ubiquitination and subsequent proteasomal degradation
of the αsubunit. HIF-1αis also hydroxylated by
factor inhibiting HIF (FIH) at a C-terminal trans-
criptional activation domain asparagine residue (Asn803)
to further hinder the transcriptional activation of HIF
in normoxic conditions through inhibition of binding
with coactivator cyclic AMP response element-binding
protein (CBP)/p300 (Mahon et al.2001).Therefore,
O2-dependent hydroxylation serves to repress HIF-1α
under normoxic conditions.
The hydroxylation of HIF-1αby PHD and FIH
dioxygenases is dependent on the availability of both
O2and co-factors 2-oxoglutarate, Fe (II) and ascorbate
(Knowles et al.2003;Pageet al.2008).Accordingly,the
hydroxylation of HIF-1αis suppressed under conditions
of hypoxia, resulting in the HIF-1αsubunit becoming
stabilised and accumulating within the cytoplasm when
acellbecomesdeprivedofanadequateO
2supply.
Accumulated HIF-1αsubsequently translocates to the
nucleus where it dimerises with HIF-1β/ARNT (Wang
et al.1995).BindingoftheHIFheterodimertohypoxia
responsive elements (HRE) within the promoter/enhancer
regions of target genes initiates the transcription of those
genes which promote adaptation to hypoxia (Wang et al.
1995; Semenza, 2011).
HIF-1-dependent effect on glycolytic flux. Amongst the
adaptive responses mediated by HIF-1 to promote cell
survival under hypoxic conditions is the HIF-1-dependent
reprogramming of glucose metabolism to reduce reliance
on O2-dependent energy production (Papandreou
et al.2006).Thisreprogrammingisachievedbythe
HIF-1-dependent upregulation of genes encoding glucose
transporters (e.g. GLUT1 and GLUT 3) and enzymes of
the glycolytic pathway (Semenza et al.1994;Ebertet al.
1995; Iyer et al. 1998; Seagroves et al. 2001), as well as
the HIF-1 mediated suppression of OXPHOS to further
promote an increased glycolytic flux (Kim et al.2006;
Papandreou et al. 2006). The increased glucose uptake
and its subsequent metabolism under conditions of hypo-
xia help maintain cellular ATP production concommitent
with reduced oxidative metabolism.
The upregulation of glucose transporters and glycolytic
enzymes is mediated by HIF-1 binding to the
hypoxia-responsive element (HRE) consensus sequence
(5"-(A/G)CGTG-3")withinthepromoterregionofthe
genes encoding these proteins to promote their increased
expression (Semenza et al. 1996). The genes encoding
glycolytic enzymes PFK-liver type (PFKL), aldolase
(ALDA), phosphoglycerate kinase-1 (PGK1), enolase
(ENOL) and lactate dehydrogenase-A (LDHA) contain
HIF-1 binding sites in their enhancer regions and are
therefore directly upregulated by HIF in response to hypo-
xia (Semenza et al.1994).Interestingly,theconsensus
sequence central to mediating HIF binding present in
human ALDA and human PGK1 are evolutionarily
conserved between humans and mice, suggesting that the
hypoxia-induced, HIF-1-dependent upregulation of these
genes is of central importance to mammalian glycolytic
regulation (Semenza et al.1994).
In addition to the HIF-1-dependent upregulation
of glycolytic enzymes in response to hypoxia, HIF-1α
can suppress OXPHOS under hypoxic conditions by
reducing metabolite entry into the TCA cycle via the
HIF-1α-dependent induction of pyruvate dehydrogenase
kinase 1 (PDK1) (Kim et al. 2006; Papandreou et al. 2006)
and LDHA (Yang et al. 2014a). PDK1 phosphorylates
the pyruvate dehydrogenase (PDH) complex, preventing
pyruvate from being converted to acetyl Co-A, thus
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Figure 2. Schematic comparing and contrasting the steady state, Warburg and Pasteur effects
In the presence of sufficient levels of oxygen, healthy cells (A) generate cellular ATP primarily by oxidative
phosphorylation by shuttling the majority of pyruvate generated (indicated by continuous arrow) into the
mitochondria. In the absence of sufficient levels of oxygen, cells reprogramme their metabolism to use glycolysis
as their primary metabolic strategy for generation of ATP (Pasteur effect). The pyruvate generated from glycolysis
is fermented into lactate and transported out of the cells via the actions of the monocarboxylate transporters
(MCTs) located within the cell membrane. Malignant cells (B)canreprogrammetheirmetabolismtopreferentially
use glycolysis as their primary metabolic strategy for energy metabolism even in the presence of sufficient levels
of oxygen and functioning mitochondria (Warburg effect). The pyruvate generated is primarily oxidised to lactate
(continuous arrow), with only a small amount of lactate being transported to the mitochondria for oxidative
phosphorylation (dashed arrow).
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6 S. J. Kierans and C. T. Taylor J Physiol 00.0
hindering the initiation of the TCA cycle. HIF-1α
also activates transcription of LDHA, which catalyses
the conversion of pyruvate generated from glycolytic
metabolism to lactate and concurrently generates NAD+,
an cofactor essential in permitting continued glycolytic
activity (Semenza et al. 1996). Together both PDK1
and LDHA divert pyruvate away from the TCA cycle
by decreasing acetyl Co-A generation from pyruvate
and instead increasing lactate production. The lactate
produced from pyruvate can then be removed from the cell
via the actions of plasma membrane transporter, mono-
carboxylate transport 4 (MCT4) to avoid competitive
inhibition of LDHA (Ullah et al.2006).
HIF-1αalso regulates OXPHOS capacity through
directly altering mitochondrial activity. HIF-1αinduces
mitochondrial autophagy in response to hypoxia thereby
conferring a protective effect in the hypoxic cell by
reducing the levels of reactive oxygen species (ROS)
generated (Zhang et al.2008).Furthermore,hypoxia
reduces respiratory capacity by regulating cytochrome
oxidase subunit expression in a HIF-1-dependent manner
to limit ROS generation under hypoxic conditions, the reby
promoting the evasion of oxidative stress-induced cell
death (Fukuda et al.2007).
Pharmacological hydroxylase inhibition and glycolysis.
In addition to the stabilisation and subsequent activation
of HIF-1αin response to hypoxia, HIF can also
be pharmacologically activated using molecules which
inhibit the actions of the PHD enzymes, such as iron
chelators (e.g. deferoxamine, DFO) (Wang & Semenza,
1993), or molecules which compete for 2-oxoglutarate
(Mole et al.2003).ModulationofPHDenzyme
activity is beneficial in promoting anti-inflammatory
responses by selectively inducing cell death in mono-
cytes (Crifo et al.2019),neutrophils(Manresaet al.
2019), and enhancing intestinal epithelial barrier function
(Cummins et al. 2008), in pro-angiogenic responses
via the HIF-dependent upregulation of vascular end-
othelial growth factor (VEGF), offering protection in
ischaemic disease (Milkiewicz et al.2004),aswellas
helping ameliorate anaemia in patients with kidney disease
by stimulating erythropoietin (EPO) production and
regulating iron absorption and metabolism (Chen et al.
2019b,c).
In the context of glucose metabolism, hydro-
xylase inhibition protects alveolar epithelial cells from
bioenergetic failure and cell death in both in vitro
and in vivo models by enhancing glycolytic metabolism
through HIF-1α(Tojo et al. 2018). Similarly, hydro-
xylase inhibition is cytoprotective in the renal proximal
tubular epithelial cell line HK2 in an oxygen-glucose
deprivation (OGD) in vitro model of ischaemia, through
upregulation of the glycolytic pathway (Ito et al.2020).
While these observations suggest that promoting glycolysis
at the cellular level through pharmacological activation
of the HIF pathway is cytoprotective, at the level of
the organism, sustained activation of the HIF pathway
arising from loss of function mutations of VHL results
in a reduced mitochondrial respiratory capacity, reduced
exercise capacity and greater blood lactate concentration
in these patients through upregulation of HIF-dependent
glycolytic genes and OXPHOS suppression under
normoxic conditions (Formenti et al. 2010; Perrotta
et al. 2020). While such findings emphasise that HIF
plays a prominent role in regulating glucose metabolism,
they also demonstrate that promoting glycolysis, for
example, through hydroxylase inhibition, may be rather
deleterious to the organism on an integrative level. The
effect of pharmacological stabilisation of HIF on the
whole organism is therefore an area worthy of further
investigation.
HIF-2αdoes not regulate glycolytic flux. HIF-2α,a
paralogue of HIF-1α,isalsonegativelyregulatedby
prolyl and asparaginyl hydroxylation (O’Rourke et al.
1999). While HIF-1αis ubiquitously expressed in all
cell types, HIF-2αexpression is cell-type specific, with
HIF-2αmRNA having been reported in vascular end-
othelial cells, kidney and gastrointestinal tract epithelial
cells, liver parenchymal cells and type II pneumocytes
(Wiesener et al. 2003). Under hypoxic conditions, HIF-2α
dimerises with constitutively expressed HIF-1βto regulate
the transcription of numerous target genes. Despite the
similarities between HIF-1αand HIF-2αin terms of
sequence homology, stabilisation and HRE recognition,
HIF-1αand HIF-2αtarget genes can differ (Tian
et al. 1997). Processes such as erythropoiesis (Gruber
et al. 2007; Rankin et al.2007)ironabsorptionand
metabolism (Mastrogiannaki et al. 2009) and carotid body
development and function (Hodson et al. 2016; Macias
et al.2018)arelargelydependentonHIF-2αexpression,
while the expression of genes encoding glucose transporter
GLUT1 and VEGF appear to be regulated by both HIF-1α
and HIF-2α(Hu et al.2003,2007).WhileHIF-1αand
HIF-2αregulated genes can overlap, multiple studies have
reported that glycolytic genes are exclusively regulated by
HIF-1α(Hu et al. 2003, 2007; Wang et al.2005).Severe
hypoxic treatment (0.1% O2)failstoinduceglycolyticgene
expression (LDHA, PGK-1, PGM-1 and PKM) in human
renal carcinoma cells (786-O) overexpressing VHL (786-O
WT-8) despite the induction of HIF-2αresponsive genes at
the same oxygen tension (Hu et al.2003).Thesefindings
support the necessary and selective role for HIF-1αin
mediating the Pasteur effect (Seagroves et al.2001).
Take n t o ge t h er, it i s e v i d e nt t h at t h e e ff e c t s m e di a ted
by HIF-1αin promoting glycolytic flux and reducing
OXPHOS capacity play a pivotal role in promoting cellular
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J Physiol 00.0 HIF-dependent regulation of glycolysis 7
adaptation to hypoxia. Given that this response can be
both beneficial in overcoming a hypoxic insult, while
also having the capacity to be exploited in the context of
cancer cell survival and tumour growth (Warburg effect),
acompleteunderstandingoftheregulationofglycolysis
under hypoxic conditions is of crucial importance.
While HIF plays a key role in mediating this adaptive
response, the involvement of other mechanisms and/or
signalling pathways in complementing the HIF-dependent
regulation of metabolic reprogramming as evident in
hypoxia must not be excluded. Such mechanisms,
including the involvement of epigenetics (Krieg et al.
2010), post-translation modifications (Agbor et al. 2011)
as well as the spatial reorganisation of glycolytic enzymes
under hypoxic conditions (Agbor et al.2011;Jinet al.
2017) are emerging topics of interest, and may explain,
at least in part, how loss of HIF-1βdoes not perturb the
glycolytic phenotype (Troy et al.2005).However,these
HIF-independent mechanisms are beyond the scope of
this review.
Hypoxia-induced glycolytic flux and the PI3K/Akt
pathway. The regulation of glycolytic flux by HIF
can also be influenced by activity of other pathways.
The phosphatidylinositol-4,5-bisphophate 3-kinase
(PI3K)/protein kinase B (Akt) pathway is a highly
conserved signalling pathway which plays a key role
in regulating cell cycle progression in response to
extracellular signals (Burgering & Coffer, 1995; Franke
et al.1995;Kohnet al.1995).Inadditiontoitsrole
in promoting cellular survival, the PI3K pathway has
been implicated in regulating HIF-1αexpression and
transcriptional activity under hypoxic conditions in a
cell-type specific manner (Alvarez-Tejado et al.2001;
Arsham et al.2002;Liuet al. 2019). In oesophageal
carcinoma cell lines, hypoxic exposure results in an
increased level of HIF-1αprotein stabilisation, glycolytic
enzyme expression and extracellular lactate production
(Zeng et al. 2016). This response was successfully reversed
using the PI3K inhibitor Wortmannin, suggesting that
the upregulation of glycolysis in response to hypoxia
in these cells was at least partially attributed to PI3K
upregulation. Additionally, PTEN inactivation mutations,
as evident in cancers, also appear to potentiate the effect
of hypoxia on HIF-dependent gene expression through
downstream effector mammalian target of rapamycin
complex 1 (mTORC1) activation (Zhong et al. 2000) while
exhibiting little to no effect on glycolytic gene expression
under normoxic conditions, supporting the idea that the
PI3K pathway may play a role in the regulation of hypoxic
glucose metabolism (Zundel et al.2000).
However, while these findings suggest there may be a
role for the PI3K signalling pathway in regulating glucose
metabolism in response to hypoxia through HIF-1α
stabilisation, the extent to which the PI3K/Akt signalling
pathway is involved in HIF-dependent, hypoxia-induced
glucose metabolism is an area which requires further
investigation. Under normoxic conditions, for example,
PI3K/Akt activation in response to cytokine stimulation
has been shown to regulate glucose uptake via the direct
regulation of GLUT1 activity and localisation (Wieman
et al. 2007). Additionally, PI3K has been shown to regulate
normoxic glucose metabolism through its regulation of the
expression of rate limiting enzymes of glycolysis HK2 and
PFK (DeBerardinis et al.2008;Xieet al.2019),exertinga
direct influence on glucose metabolism without activating
mitochondrial OXPHOS (Elstrom et al. 2004). Such
findings suggest that the effects of the PI3K/Akt signalling
pathway on glucose metabolism are not confined to those
mechanisms which rely on HIF activity. Understanding
the potential crosstalk and complementarity between
HIF-dependent and HIF-independent mechanisms of
hypoxic glucose metabolic regulation is an area of key
importance from a physiological standpoint.
Hypoxia-induced glycolytic flux and non-coding RNAs
(ncRNA). RNAs, including coding RNAs such as
messenger RNA (mRNA) and non-coding RNAs such as
microRNA (miRNA), are heavily regulated by hypoxia in
a HIF-dependent manner (Schodel et al.2011;Choudhry
et al. 2014). Among the non-coding RNAs regulated by
hypoxia (reviewed by Shen et al.2013),longnon-coding
RNAs (lncRNAs) have emerged as a new class of trans-
cript commonly regulated by hypoxia (Choudhry et al.
2014; Choudhry & Harris, 2018). Interestingly, a number
of lncRNAs have been implicated in the regulation of
hypoxia-enhanced glucose metabolism.
Long non-coding RNAs (lncRNAs) are a large class
of heterogenous regulatory transcripts, greater than 200
nucleotides in length, which lack evidence of protein
coding potential (Rinn & Chang, 2012). Of those lncRNAs
regulated by hypoxia, there is evidence to suggest hypoxic
regulation is mediated by the binding of HIF to an HRE
consensus sequence located within the promoter region
of these lncRNAs (Choudhry et al. 2014; Choudhry &
Harris, 2018). LncRNAs lincRNA-p21, lncHIFCAR and
lncRNA-UCA1 have all been implicated in regulating the
HIF-dependent increase in glycolysis as evident under
hypoxic conditions (Li et al. 2014; Xue et al. 2014; Yang
et al.2014b;Shihet al.2017).
Long intragenic non-coding RNA (lincRNA)-p21
(lincRNA-p21) has been implicated as a critical mediator
of the hypoxia-dependent increase in glycolysis via its
role in promoting HIF-1αstabilisation (Yang et al.
2014b). LincRNA-p21, a key repressor of p53-dependent
non-transcriptional responses (Huarte et al. 2010),
is strongly induced by hypoxia in a dose- and
time-dependent manner through the binding of HIF-1αto
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8 S. J. Kierans and C. T. Taylor J Physiol 00.0
an HRE consensus sequence located within the promoter
region of lincRNA-p21 in mammalian cells (Yang et al.
2014b). LincRNA-p21 induction increases the expression
of glycolytic enzymes LDHA and GLUT1 to result in
an increased glucose uptake and lactate production
within these cells in response to O2deprivation. Inter-
estingly, in addition to the regulation of lincRNA-p21 by
HIF-1α, lincRNA-p21 can reciprocally regulate HIF-1α
levels under hypoxic conditions by interfering with the
HIF-1α-VHL interaction, thereby preventing HIF-1α
proteasomal degradation (Yang et al.2014b).
In addition to the regulation of metabolic
reprogramming by lincRNA-p21 in response to
hypoxia, long non-coding RNA HIF-1αco-activating
RNA (lncHIFCAR) has also been implicated in regulating
hypoxia-induced glucose uptake and lactate production
in oral squamous cell carcinoma cell lines (Shih et al.
2017). LncHIFCAR is induced by hypoxic stress and is
required for the hypoxia-induced activation of HIF-1
target genes, glycolytic enzymes LDHA and PDK1 (Shih
et al.2017).Theupregulationoftheseglycolyticenzymes
is achieved via the lncHIFCAR-dependent augmentation
of the transcriptional activity of HIF-1αby lncHIFCAR
associating with the chromatin loci of HIF-1 target
proteins (Shih et al.2017).
Finally, lncRNA urothelial associated carcinoma 1
(UCA1) has also been implicated as a hypoxia-inducible
lncRNA (Xue et al.2014),whichplaysapivotalrole
in accelerating glycolysis in bladder cancer cells via the
upregulation of the HIF-1-dependent glycolytic enzyme
hexokinase 2 (HK2) (Li et al.2014).Interestingly,while
the downstream effects of HIF-dependent upregulation
of lincRNA-p21 and lncHIFCAR appear to be mediated
primarily by the HIF pathway, lncRNA-UCA1 can
mechanistically regulate glycolytic gene expression via the
mammalian target of rapamycin (mTOR) pathway (Li
et al.2014).Therefore,thisobservationfurtherhighlights
the potentially complementary nature of HIF-dependent
and HIF-independent regulation of glucose metabolism
under hypoxic conditions. However, while these findings
outline a likely role of non-coding RNAs in regulating
HIF-1-dependent reprogramming of glucose metabolism,
as evident in hypoxic conditions, the mechanisms under-
lying the regulation of lncRNAs in both normoxic and
hypoxic conditions remain to be fully elucidated.
Functional physiological consequence of a
hypoxia-induced increase in glycolysis
The hypoxia-induced enhancement of glycolysis promotes
adaptation to O2deprivation by supporting ATP
production following suppression of OXPHOS. However,
the functional consequence of an enhanced glycolytic
flux extends beyond that of provision of bioenergetic
energy equivalents in the form of ATP. These consequences
include both the regulation of the immunity and
neovascularisation, and play important roles in the body’s
adaptation to hypoxia. The implications of increased
glycolytic flux from a physiological aspect are outlined
below and highlight our crucial need to understand the
regulation of cellular glucose metabolism under hypoxic
conditions in its entirety.
Endothelial cells. Endothelial cells (ECs) are highly
plastic cells which form the inner lining of both blood and
lymphatic vessels. Despite their close proximity to a rich
oxygen supply in the blood, glycolysis is the predominant
bioenergetic pathway for ECs, generating up to 85% of the
cellular ATP content in arterial, venous, lymphatic and
microvascular ECs (Culic et al.1997;DeBocket al. 2013).
ECs can remain quiescent for years, only becoming active
during periods of O2/nutrient deprivation, in response
to tissue damage or as a result of pathological conditions
such as inflammation or cancer (Vandekeere et al.2015).
Under such conditions, ECs can rapidly reprogramme
their metabolism to double their metabolic rate via the
further upregulation of the glycolytic pathway through
phosphofructokinase-2/fructose-2,6-bisphosphatase-3
(PFKFB3) (Dobrina & Rossi, 1983). This increase in
glycolytic metabolism promotes angiogenesis at the
vascular forefront via endothelial tip cell PFKFB3, which
when silenced, impairs vessel sprouting (De Bock et al.
2013). Endothelial tip cell extension is followed by
endothelial stalk cell proliferation to promote elongation
of the vessel. The newly formed vessel then becomes lined
with is endothelial phalanx cells, with a lower (albeit, still
substantial) glycolytic rate than the actively proliferating
ECs, allowing for vessel perfusion (De Bock et al.2013).
While it seems counterintuitive for ECs to rely
on glycolytic metabolism as their primary metabolic
strategy in both quiescent and active states, there
are multiple benefits derived from this preferential
mechanism of energy metabolism. A particular benefit
in the context of ECs of relying on anaerobic glycolysis as
ameansofenergyproductionisinthevascularisation
of tissues. In the absence of O2,glycolyticECscan
continue to promote angiogenesis in the absence of
O2in attempts to improve overall tissue oxygenation.
Inhibiting the glycolytic pathway using glucose mimetic
2-deoxy-D-glucose (2-DG) inhibits angiogenesis in both
in vitro and in vivo models by interfering with
N-linked glycosylation (Merchan et al.2010),highlighting
a pertinent role for glycolysis in neovascularisation.
Glycolytic metabolism is also beneficial for ECs, as
lactate, the end product of glycolysis, functions as a
pro-angiogenic signalling molecule in highly glycolytic
ECs via the activation of the NFκB/CXCL8 pathway and
the stabilisation of HIF-1α(Dobrina & Rossi, 1983; Vegran
et al. 2011; Sonveaux et al.2012;Eelenet al.2018).
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J Physiol 00.0 HIF-dependent regulation of glycolysis 9
The glycolytic pathway can also beneficially provide
numerous co-factors which can be diverted to
biosynthetic growth pathways to support anabolic
cell growth and proliferation. Glycolytic intermediates
glucose-6-phosphate and 3-phosphoglycerate, for
example, are supplied to the pentose phosphate and
serine biosynthetic pathways, respectively, where they
are used in the production of ribose and amino acids
which both play important roles in supporting cell
growth (Horecker, 2002). In addition to the low level
of OXPHOS evident in quiescent and active ECs, it has
been noted that angiogenesis is not affected by complete
inhibition of mitochondrial respiration (De Bock et al.
2013). A low level of OXPHOS generates fewer reactive
oxygen species (ROS), subjecting ECs to a reduced level of
oxidative stress despite their proximity to a high-oxygen
environment, therefore protecting ECs from ROS-induced
cell death.
Take n t o ge t h er, th e s e ob s e r v at i o ns s u g ge s t th a t w h i l e
glycolysis may not be the most efficient way of producing
ATP, EC s d erive be n e fi t f r o m g lycoly t i c metab o l i s m in
terms of angiogenic potential, with glycolysis playing a
pivotal role in promoting EC proliferation and motility
(De Bock et al.2013).Whilebeingbeneficialfrom
aphysiologicalstandpointinpromotingadaptationto
O2deprivation, the angiogenic potential of ECs can be
detrimental in pathophysiological states such as cancer
development and progression. In such situations, the
hypoxic core of tumours can result in the inappropriate
initiation of angiogenesis and adversely promote tumour
growth and survival. However, the identification of
PFKFB3 as the primary driver of glycolytic in ECs
has been of great utility as small molecule compound
3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-1-one (3PO),
an inhibitor of PFKFB3, can reduce proliferation and
migration of ECs in both in vitro and in vivo models
(De Bock et al. 2013). This finding suggests that
pharmacological intervention in the glycolytic pathway
may present new opportunities for anti-angiogenic
therapies.
Immune cells. In addition to the implications of an
enhanced glycolytic flux for endothelial cell biology,
glycolytic metabolism plays a regulatory role in immune
cell function. A variety of immune cells, including
dendritic cells (Krawczyk et al. 2010), classically activated
M1 macrophages (Michl et al. 1976), neutrophils (Sbarra
& Karnovsky, 1959), lymphocytes (T cells (Frauwirth et al.
2002; Wang et al. 2011) and B cells (Doughty et al. 2006))
adopt a high-glycolytic phenotype once activated. This
high-glycolytic phenotype therefore confers a particular
advantage to immune cells in allowing for mounting of
an immune response in tissues deprived of an adequate
oxygen supply. Such tissues include those which are
inflamed, infected or tumour burdened, where hypo-
xia is common place, as well as those tissues which
experience persistent low levels of oxygenation (so called
‘physiological hypoxia’), such as the bone marrow,
placenta or gastrointestinal tract (Taylor & Colgan,
2017).
Immune cell metabolism and the tissue micro-
environment. Unlike cancer cells, whereby an increased
rate of glycolysis and reduced reliance on OXPHOS often
arises from genetic aberrations, an enhanced glycolytic
flux in immune cells is not permanent and, instead,
immune cells obtain the capacity to rapidly alter their
metabolic profile in response to external signals (Ganeshan
& Chawla, 2014). Such external signals include hypo-
xia which is commonly encountered by immune cells in
both physiological and pathophysiological settings. The
activation of glycolysis in immune cells is regulated by
HIF-1αin activated M1 macrophages, dendritic cells,
lymphocytes and neutrophils, and co-regulated by HIF-1α
and PI3K/Akt pathway in dendritic cells and CD4+Tcells
(Ganeshan & Chawla, 2014; Colgan et al.2020).
The metabolic reprogramming of immune cells
following their activation has multiple effects on
immune responses. In dendritic cells (DCs), metabolic
reprogramming to a high-glycolytic phenotype is
necessary for DC activation and regulates DC cytokine
production and antigen presenting capability (Krawczyk
et al.2010;Evertset al.2014).Inactivatedneutrophils
and classically activated macrophages, glycolysis is crucial
to support their infiltrating capacity and microbicidal
actions (Valentine & Beck, 1951; Sbarra & Karnovsky,
1959), such that inhibition of glycolysis with 2-DG pre-
vents macrophage activation and inhibits endocytosis of
complement 3 (C3) and immunoglobulin (IgG) bound
particles (Michl et al.1976;Boxeret al.1977).Inhibition
of glycolysis hinders the development of T cells into
TH17 cells, whilst promoting the production of regulatory
T(T
reg) cells, suggesting that the glycolytic pathway is
an important mediator of T cell differentiation (Shi
et al.2011).InactivatedCD4
+Tcells,glycolysisplays
aprimaryroleinpromotingtheactivationofeffector
T cells (Michalek et al.2011),anddownregulating
glycolysis by silencing of PFKFB3 in CD4 T cells renders
these cells highly susceptible to apoptosis and, counter-
intuitively, impairs autophagy (Yang et al.2013).Finally,
sustained glycolytic metabolism following the cessation of
an immune response impairs the ability of CD8+Tcells
to form long-term immune memory such that inhibiting
glycolytic flux preserves the formation of memory CD8+T
cells (Sukumar et al.2013).Hypoxiacanthereforegreatly
directly influence immune cell phenotype and function
via metabolic reprogramming to favour a glycolytic
phenotype.
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10 S. J. Kierans and C. T. Taylor J Physiol 00.0
Immune cell metabolism and the tumour micro-
environment. While physiological hypoxia can promote
immune cell homeostasis under hypoxic conditions by
altering immune cell metabolic status, pathological hypo-
xia, as evident in the core of tumours can regulate immune
cell effector function which in turn plays a role in tumour
development (Lee et al.2010).Numerousimmunecell
types are recruited to a tumour to reside within the
tumour microenvironment. These include those immune
cells important for mediating anti-tumour effects such
as DCs and natural killer cells, as well as those which
promote pro-tumour effects, such as tumour-associated
macrophages (TAMs) and myeloid-derived suppressor
cells (MDSCs) (Reviewed by Ou & Lv, 2020).
Hypoxia within the tumour microenvironment
promotes tumour growth by numerous mechanisms.
First, hypoxia within the tumour microenvironment can
accelerate the rate of glycolysis in MDSCs via HIF-1α,to
promote proliferation of these cells and potentiate their
immunosuppressive effects (Corzo et al. 2010). MDSCs
can exert immunosuppressive effects by suppressing
T-cell responses and modulating cytokine production
from phagocytic macrophages to promote immuno-
evasion (reviewed by Millrud et al. 2017). Hypoxia
within the tumour microenvironment further promotes
the differentiation of monocytic MDSCs (M-MDSCs)
into TAMs via HIF-1α(Corzo et al.2010).Interestingly,
TAMs within the mi c r o e nvironment of b r e a s t c ancers
upregulate aerobic glycolysis of tumour cells through
shuttling of extracellular vesicles containing lncRNA
HIF-1α-stabilising non-coding RNA (HISLA), which
promotes HIF-1αstabilisation by preventing the inter-
action between HIF-1αand the PHD enzymes (Chen et al.
2019a). These highly glycolytic tumour cells positively
regulate HISLA levels in TAMs via an increased lactate
production to further promote tumour cell glycolytic
activity and contribute to the immunoevasive tumour
phenotype (Chen et al.2019a).
Secondly, highly glycolytic tumour cells suppress T
cell activity by depriving immune cells of an adequate
glucose supply (Chang et al.2015),withlowlevelsof
glucose resulting in T cell hyporesponsiveness, even when
tumours are highly antigenic (Maciver et al.2008).Glucose
restrictions within the tumour microenvironment as a
result of increased glucose uptake of hypoxic tumour cells
can also favour the activation of an M2-like phenotype
in tumour infiltrating macrophages, thereby promoting
an anti-inflammatory response and promoting tumour
growth (Chang et al.2015).
Hypoxic tumours also have a high concentration of the
glycolytic end product lactate in their microenvironment
as a result of an increased glycolytic flux in tumour cells.
High levels of lactate in the tumour microenvironment
reduce immune cell activity. Specifically, tumour derived
lactate inhibits T cell cytokine production by disturbing
T cell metabolic homeostasis (Fischer et al. 2007; Dietl
et al. 2010). Tumour-derived lactic acid also alters the
antigen-presenting capacity and cytokine production of
human monocyte-derived DCs (Gottfried et al.2006)
and inhibits monocyte activation, migration and cyto-
kine release (Dietl et al. 2010; Goetze et al. 2011).
Lactate can also have profound effect on macrophage
polarisation, by inducing arginase 1, VEGF and other
M2-associated genes in TAMs, thereby resulting in the
development of a tumour-promoting phenotype in these
cells (Colegio et al.2014).Anincreasedintracellularlactate
concentration results in an increased level of histone
lactylation. The effect of this increased lactylation is the
increased expression of genes typical of M2-macrophage
polarisation in M1-macrophages (Zhang et al.2019).
Finally, lactate inhibits the activation and survival of
natural killer cells in both in vitro and in vivo settings
(Brand et al. 2016). To summarise, high levels of lactate
within the tumour microenvironment as a result of an
enhanced glycolytic flux can have a profound effect
on immune cell function, resulting in notable tumour
evasion.
Take n t o ge t h er, th e s e fin d i n g s h ig h li g ht t h at d i f fe r e nce s
in metabolic status, be that originating from the immune
cell itself, or indeed, the immune cell microenvironment
have a significant impact on immune cell function and can
profoundly influence an immune response. The impact of
inhibiting a high-glycolytic phenotype in immune cells
has been investigated in in vivo models of autoimmune
conditions such as experimental autoimmune neuritis
(Liu et al.2018)andsystemiclupuserythematosus(Yin
et al. 2016), and the findings suggest that inhibition
of glycolysis can have profound effects on the adaptive
and innate immune response with notable reductions
in clinical symptoms. Gaining a more thorough under-
standing of the differences in metabolic profile within a
physiological or pathological immunological niche may
lead to a method of targeted treatment for immune
pathologies or reversal of tumour cell immune evasion
in coming years.
Conclusion
In summary, studies in both in vitro and in vivo
models have demonstrated a profound capacity for hypo-
xic cells to adapt to O2deprivation by altering trans-
criptional and translational responses to promote glucose
uptake and its subsequent anaerobic catabolism. While
this response is mediated primarily by HIF stabilisation
and HIF-target gene expression, it is still possible that
alternative mechanisms are also involved in promoting
this glycolytic phenotype. The integration of these
responses and the elucidation of their precise molecular
mechanisms and crosstalk are fundamental to our
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J Physiol 00.0 HIF-dependent regulation of glycolysis 11
understanding of human physiology, and may represent
an area of important translational potential for human
disease.
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Additional information
Competing interests
The authors declare no competing interests.
Author contributions
Both authors have approved the final version of the
manuscript and agree to be accountable for all aspects
of the work. All persons designated as authors qualify for
authorship, and all those who qualify for authorship are
listed.
Funding
S.J.K. is funded by a UCD Advance PhD Course Scheme
grant awarded to C.T.T. (R19448).
Keywords
glycolysis, HIF, hypoxia, metabolism
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