J Mammary Gland Biol Neoplasia

DOI 10.1007/s10911-013-9310-8

Biology of Glucose Transport in the Mammary Gland

Feng-Qi Zhao

Received: 19 August 2013 / Accepted: 29 October 2013

# Springer Science+Business Media New York 2013

Abstract Glucose is the major precursor of lactose, which is implications for the treatment of breast cancer because glucose
synthesized in Golgi vesicles of mammary secretory alveolar uptake and GLUT expression are up-regulated in breast cancer
epithelial cells during lactation. Glucose is taken up by mam- cells to accommodate the increased glucose need.
mary epithelial cells through a passive, facilitative process,
which is driven by the downward glucose concentration gra- Keywords Glucose . Glucose transport . GLUT . Lactose
dient across the plasma membrane. This process is mediated synthesis . Mammary gland . Regulation
by facilitative glucose transporters (GLUTs), of which there
are 14 known isoforms. Mammary glands mainly express Abbreviations
GLUT1 and GLUT8, and GLUT1 is the predominant isoform
with a K m of ~10 mM and transport activity for mannose and 2-DG 2-deoxy-D-glucose
galactose in addition to glucose. Mammary glucose transport 3-O-MG 3-0-methyl-D-glucose
activity increases dramatically from the virgin state to the -LA -lactalbumin
lactation state, with a concomitant increase in GLUT expres- 4Gal-T1 1,4-galactosyltransferase
sion. The increased GLUT expression during lactogenesis is G6P Glucose-6-phosphate
not stimulated by the accepted lactogenic hormones. New GLUT Facilitative glucose transporter
evidence indicates that a possible low oxygen tension MBF Mammary blood flow
resulting from increased metabolic rate and oxygen consump- MECs Mammary alveolar epithelial cells
tion may play a major role in stimulating glucose uptake and NADPH Nicotinamide adenine dinucleotide phosphate
GLUT1 expression in mammary epithelial cells during SGLT Na+/glucose cotransporter
lactogenesis. In addition to its primary presence on the plasma
membrane, GLUT1 is also expressed on the Golgi membrane
of mammary epithelial cells and is likely involved in facilitat-
ing the uptake of glucose and galactose to the site of lactose Introduction
synthesis. Because lactose synthesis dictates milk volume,
regulation of GLUT expression and trafficking represents Glucose, a universal and essential fuel in energy metabolism
potentially fruitful areas for further research in dairy produc- and in the synthesis pathways of all mammalian cells [1], is
tion. In addition, this research will have pathological constantly required at sufficient levels in the blood stream. In
lactating animals, glucose is the major precursor for lactose
and is a substrate for the synthesis of milk proteins and fat in
F.<Q. Zhao (*)
mammary secretory (alveolar) epithelial cells (MECs). The
Laboratory of Lactation and Metabolic Physiology, Department of
Animal Science, University of Vermont, 211 Terrill Building, 570 mammary gland cannot synthesize glucose from other precur-
Main Street, Burlington, VT 05405, USA sors due to its lack of glucose-6-phosphatase [2, 3]. Therefore,
e-mail: fzhao@uvm.edu it is dependent on the blood supply for its glucose needs. For a
dairy cattle with an average milk production of 40 kg/day, up
F.<Q. Zhao
Institute of Dairy Science, College of Animal Sciences, Zhejiang to 3 kg of glucose must be taken up daily from the blood by
University, Hangzhou, Zhejiang 310029, China the mammary gland [4, 5]. The supply of glucose to the

mammary gland is a metabolic priority in lactating mammals
[6]. Glucose uptake by the mammary gland can account for as
much as 6085 % of the total glucose that enters the blood [5,
7]. Because lactose maintains the osmolarity of milk and thus,
the rate of lactose synthesis serves as a major control of milk
volume [8, 9], glucose uptake by the mammary gland is
considered a rate-limiting step in milk production [4, 10].
Mammary glucose uptake is a passive and facilitated trans-
port process, driven by a more than 10-fold inward glucose
concentration gradient across the plasma membrane of MECs
[1116]. This review will focus on our current understanding
of the molecular mechanisms and regulation of glucose trans-
port in MECs, with a particular emphasis on the physiological
roles of glucose transporters in supporting milk production in
dairy animals. However, the pathophysiological role of glu-
cose transport in breast cancer will also be introduced.
Glucose Utilization and Metabolism in the Mammary
Gland
In lactating mammary gland, glucose is mainly used in the
following processes: 1) lactose synthesis, 2) nicotinamide ade-
nine dinucleotide phosphate (NADPH) generation, 3) milk lipid
synthesis, 4) energy production, and 5) nucleic acid and amino
acid syntheses (Fig. 1). In lactose synthesis, glucose is first
irreversibly phosphorylated to glucose-6-phosphate (G6P) and
then converted to UDP-galactose in the cytoplasm. Both glucose
and UDP-galactose are taken up by Golgi vesicles and used to
synthesize lactose by the lactose synthase located in the Golgi
membrane. Lactose synthase consists of two polypeptide sub-
units: -lactalbumin (-LA) and 1,4-galactosyltransferase
(4Gal-T1) [17]. The mammary-specific -LA changes the
specificity of 4Gal-T1 from N-acetylglucosamine to glucose
to produce lactose [18]. Glucose-6-phosphate can also enter
either the glycolysis pathway or the pentose phosphate shunt
(Fig. 1). During glycolysis, G6P is converted to triose-phosphate
and then to pyruvate. Triose-phosphate is also a substrate for
producing -glycerol-phosphate, which provides a backbone for
the biosynthesis of triacylglycerol, the major milk lipid. Pyruvate
is actively taken up by mitochondria to produce acetyl-CoA,
which then enters the tricarboxylic acid cycle (TCA) to produce
ATP (Fig. 1). The TCA cycle substrate citrate can also leave the
mitochondria and be converted to acetyl-CoA by ATP citrate
lyase in the cytoplasm. The cytosolic acetyl-CoA is used in fatty
acid elongation. In addition, pyruvate and the TCA cycle sub-
strates can be used to generate the non-essential amino acids
(such as alanine and glutamine) used in milk protein synthesis
(Fig. 1), as demonstrated by the observation that the administra-
tion of 2-deoxy-D-glucose (2-DG), a non-metabolized glucose
analogue, inhibits both lactose synthesis and protein synthesis
and secretion in rat mammary gland [19]. The pentose phosphate
shunt produces NADPH and ribose. The first provides essential
J Mammary Gland Biol Neoplasia
reducing equivalents for fatty acid biosynthesis, and the latter is a
substrate for nucleic acid (RNA) synthesis.
In lactating goats, 75 % of the glucose taken up by the
mammary gland is used for lactose synthesis, and 4.5 %
appeared in milk triacylglycerol [20]. Animal starvation does
not affect these percentages of glucose utilization.
Approximately 8 % of glucose is completely metabolized in
the pentose phosphate shunt, which accounts for all CO2
produced from glucose and provides at least 34 % of the
NADPH required for de novo fatty acid synthesis. Little
glucose is used for fatty acid elongation. These data are
consistent with the in vitro study in lactating bovine mammary
gland [21]. In non-ruminant animals, such as sows, rats, and
humans, most of the glucose taken up by the mammary gland
is also used to synthesize lactose and milk fat glycerol
[2224]. In sows, at least 40 % of the glycerol and 59 % of
the lactose are derived from plasma glucose [24]. In humans,
98 % of the glucose and 68 % of the galactose in lactose are
derived from plasma glucose [22]. However, a striking differ-
ence in non-ruminant animals is that milk fatty acids are
extensively derived from plasma glucose [24]. In addition,
54 % of mammary CO2 originates from the oxidation of
glucose in sows, indicating that glucose is a more important
energy source in non-ruminant mammary glands.
Glucose Uptake in the Mammary Gland
Infusion studies of radiolabeled glucose have showed an
average glucose uptake of approximately 9 mg/min/100 g of
tissue in lactating sow and goat mammary glands [24, 25]. In
lactating Holstein cows, Cant et al. [26] showed a mammary
glucose uptake of approximately 300 mmol/h to support a
milk production of 0.6 kg/h. This uptake was close to an
earlier observation that 72 g of glucose is taken up by the
bovine mammary gland to produce 1 kg of milk [4]. If we
assume that each gram of mammary tissue produces 1.67 g of
milk daily, a ratio that is observed in several species [27], the
glucose uptake rate in lactating bovine mammary gland based
on the milk yields in the above two studies can be estimated to
be ~810 mg/min/100 g, which is very close to the uptake
rates observed in sows and goats. Mammary glucose uptake in
lactating ruminant animals accounts for 6085 % of the total
glucose entering the circulation [5, 7, 28].
Mammary glucose uptake is affected by many factors,
including mammary developmental stages, animal nutritional
status, and mammary metabolism. In goats, the uptake in-
creases by more than 20-fold from pregnancy to peak lactation
and then gradually declines following the decrease in milk
yield [29]. The uptake is more than doubled during the last
2 days before parturition, followed by an even more substan-
tial increase immediately after parturition [30]. The rapid and
high magnitude of increase of glucose uptake is, thus,
J Mammary Gland Biol Neoplasia

Fig. 1 Main pathways of glucose Mammary epithelial cell

utilization in mammary alveolar Cytoplasm Glutamine
epithelial cells. In mammary Mitochondia
epithelial cells, glucose is mainly Alanine
Glutamate Acetyl-CoA
ATP
used in 1) lactose synthesis in the Golgi
Golgi, 2) energy production via - NAD+ H2O
LA -KG
glycolysis and the tricarboxylic
TCA ETC
acid cycle (TCA), 3) NADPH GT Lactose Pyruvate O2
production via the pentose -Glycerol-P NADH
ADP
phosphate shunt, and 46) UDP-galactose
providing substrates for lipid Triose-P
Citrate
Glucose
(glycerol and fatty acid, 4), Ribose
protein (nonessential amino acid, Acetyl-CoA (Primer)
5) and nucleic acid (ribose, 6) Nucleic acids
syntheses. -KG, - Fructose-6-P Malonyl-CoA
ketoglutarate; -LA, - Pentose
UDP-galactose NADPH
lactoalbumin; ETC., electron phosphate
transfer chain; GLUT, facilitative shunt
glucose transporters; GT, 1,4-
galactosyltransferase; and P, Glucose-1-P Glucose-6-P NADP+
phosphate Fatty acids
Glucose -Glycerol-P
Glucose
Milk lipid
GLUT

Capillary
Glucose

considered to be an important index of the onset of copious
milk secretion [30]. During nutrient restriction, mammary
glucose uptake decreases to 25 % after 48 h of starvation
[20]. Increasing the milking intervals decreases the mammary
glucose uptake by decreasing mammary blood flow and ex-
traction rate [31, 32]. Mammary glucose uptake has been
observed to be partially dependent on mammary acetate up-
take in cows [33], indicating an interaction between these two
nutrients in the bovine mammary gland. This interaction was
also observed in an in vitro study in which the rate of glucose
oxidation in bovine mammary tissue is influenced by the
acetate concentration [21]. However, mammary glucose up-
take does not appear to be affected by amino acid availability
because an infusion of amino acids to the abomasum of cows
does not alter mammary glucose uptake [34, 35]. Mammary
glucose uptake is stimulated by growth hormone and thyrox-
ine (T4) in ruminant animals [36, 37] but not by insulin across
the species [29, 3842].
Mammary glucose uptake is a function of the mammary
glucose supply and the glucose transport activity across the
plasma membrane of MECs. The former is determined by the
artery glucose concentration and blood flow to the mammary
gland. In mammals, the arterial glucose concentration is tightly
regulated by insulin and other endocrine hormones to remain
within a narrow range. Studies in cows and goats could not find
a perceptible relationship between mammary arteriovenous
differences in glucose and arterial glucose concentrations for
various experimental conditions [26, 29, 33], indicating that the
artery glucose concentration may not be a major factor in
determining mammary glucose uptake in normal physiological
conditions. This finding is consistent with several observations
that raising plasma glucose concentrations by an infusion of
glucose to the abomasum/intestine or directly to the blood is
often not coupled with increased lactose synthesis and milk
production in cows and humans [4244], although opposite
observations have also been reported [5, 45]. Mammary blood
flow (MBF) plays an important role in regulating nutrient
supply to the mammary gland [10, 46]. Generally, there is a
close relationship between MBF and milk production [31, 47,
48]. However, increasing MBF is also not necessarily followed
by increases in milk production and lactose synthesis [5, 49,
50]. Thus, these observations, together with the steep glucose
concentration gradient across the basolateral plasma membrane
of MECs (see below), suggest that the rate of glucose transport
across the basolateral membrane of MECs serves a major
impediment to mammary glucose uptake.
Glucose Transport and Glucose Transporters
in Mammalian Cells
As a polar, hydrophilic molecule, glucose cannot pass through
the hydrophobic phospholipid bilayer of cell plasma mem-
brane through simple diffusion [51]. Glucose transport
through biological membrane requires specific mechanisms
and transporter proteins. In bacteria, glucose is translocated
through the cell membrane by multiple transporter systems,
including the phosphotransferase system (PTS) and sugar/
proton symporters [52]. In yeast, glucose transport occurs
through facilitated diffusion, mediated mainly by the family

of hexose transporters (Hxt) [53]. In mammalian cells, glucose
uptake is carried out by two independent mechanisms, medi-
ated by two families of transporter proteins [54]:
1) Na +-dependent transport: Glucose transport through
the apical membrane of epithelial cells in the intestine,
choroid plexus, and kidney proximal convoluted tubule
relies on a secondary active, Na+-linked transport mech-
anism, mediated by Na+/glucose cotransporters (protein
symbol SGLT, gene symbol SLC5 for solute carrier fam-
ily 5) [5557]. This secondary active transport moves
glucose unidirectionally against its concentration gradi-
ent, using the energy provided by the cotransport of Na+
ions down their electrochemical gradient, established by
Na+/K+/ATP pumps [58]. This sodium-dependent active
transport can be inhibited by phlorizin [59, 60].
The SLC5 family currently has 12 structurally related
members (SGLT1-6, SMIT1, NIS, SMVT, CH1, SMCT1,
and SMCT 2) [57]. These transporters have various tissue
distributions and substrate specificities. Of these trans-
porters, SGLT1(SLC5A1 ), 2(SLC5A2 ), 3(SLC5A4 ),
4(SLC5A9), 5(SLC5A10), and SMIT1 (SLC5A3) have
shown transport activity for glucose, while the others
have predominant substrate specificities to anions, vita-
mins, and short chain fatty acids [57]. In addition to these
transport activities, SGLTs can function as water and urea
channels [61, 62] and as a glucose sensor [63]. The
physiological roles of SGLT1 and 2 are relatively well
characterized. SGLT1 is primarily expressed in the brush
border membrane of mature enterocytes in the small
intestine and plays a major role in the absorption of
dietary glucose and galactose from the intestinal lumen
[56]. SGLT2 is predominantly expressed in the brush
border membrane of renal proximal convoluted tubule
epithelial cells and is involved in glucose reabsorption
from the glomerular filtrate during urine production [64].
2) Facilitated transport : Glucose transport across the
basolateral membrane of epithelial cells in the intestine,
choroid plexus, and kidney proximal convoluted tubule
and across the plasma membrane of almost all other cells
is a facilitated diffusion process, mediated by facilitative
glucose transporters (protein symbol GLUT, gene symbol
SLC2). During facilitated diffusion, glucose is bidirec-
tionally transported down its concentration gradient with-
out requiring energy (ATP). The GLUT-mediated process
is specifically inhibitable by cytochalasin-B or phloretin
[6567].
The GLUT family belongs to one of two largest fam-
ilies of membrane transporters known as the major facil-
itator superfamily (MFS), which includes bacterial sugar/
proton symporters, yeast Hxt transporters, and plant
hexose/proton symporters [68, 69]. There are 14 mamma-
lian GLUT proteins, named GLUT1-12 and 14 and HMIT
J Mammary Gland Biol Neoplasia
[70]. Although these proteins are structurally conserved
and share the same structure characteristics [55], each
member has specific substrate specificities, tissue distri-
butions and regulatory properties, which are reviewed
elsewhere [55, 70]. Here, I only briefly summarize the
currently known major functions of the relatively well-
studied individual GLUTs. GLUT1 is ubiquitously
expressed in many tissues and cells and plays a major role
in cell and tissue basal glucose uptake and glucose trans-
port across the tissue-blood barrier [71, 72]. GLUT2 is a
low-affinity transporter for glucose (K m ~17 mM) and
other monosaccharides [73]. It is involved in glucose
uptake and release in hepatocytes [74, 75] and in the
absorptive epithelial cells of the intestine and the kidney
convoluted tubule [76, 77] and is also involved in glucose-
sensing in pancreatic -cells, the liver, and the hypothal-
amus [7881]. GLUT3 has a high affinity for glucose [73],
and its high expression in the brain facilitates the high-
priority glucose utilization in this tissue [82, 83]. GLUT4
is one of the most studied GLUTs because of its sensitivity
to insulin. In adipose and muscle cells, insulin stimulates
its translocation from intracellular pools to the plasma
membrane to take up glucose and lower blood sugar levels
[84]. This regulation is jeopardized in the type II-diabetes
patients [85]. GLUT5 is a fructose transporter and shows
no transport activity for glucose [86]. It is involved in
fructose uptake from the intestinal lumen [86, 87].
GLUT8 is a high-affinity glucose transporter (K m ~
2 mM) and is expressed most abundantly in testes [88,
89]. It may thus play a major role in the provision of
glucose to mature spermatozoa. GLUT12 is up-regulated
in breast ductal cell carcinoma, implying a role in breast
tumorigenesis [90], but it shows low glucose transport
activity when expressed in Xenopus oocytes [91].
Recently, a new class of glucose transporters, named
SWEETs, has been identified by the Frommer group [92,
93]. The SWEET family of transporters is conserved in bacte-
ria, plants, and animals/humans and is not structurally related
to either the GLUT or SGLT family. The eukaryotic SWEET
proteins are predicted to have 7 transmembrane domains,
unlike the 12 and 14 domains of GLUTs and SGLTs, respec-
tively [55]. The activity of a glucose uniporter has been dem-
onstrated in several plant and animal/human SWEETs, and
sucrose transport activities have been reported for several plant
SWEETs [92, 93]. The Arabidopsis SWEET1 has a low affin-
ity for glucose (K m ~9 mM) and does not appear to transport
mannose, fructose, or galactose [92]. The single human homo-
log (HsSWEET1) does not show significant glucose uptake
activity when expressed in yeast and oocytes but has weak
efflux activity in oocytes. HsSWEET1 is ubiquitously
expressed in human tissues and cells, with the highest expres-
sion in the oviduct, epididymis, and intestine. HsSWEET1
J Mammary Gland Biol Neoplasia
expression in HEK293T cells showed that it is predominantly
located in the Golgi with low expression in the plasma mem-
brane. Chen et al. [92] hypothesized that the animal/human
SWEET1 may be involved in glucose efflux from the intestine
and liver cells, but the physiological roles of SWEETs in
mammals remain uncertain.
Glucose Transport in MECs
Arteries have a physiological glucose concentration of 48 mM
in humans [94], 911 mM in rodents [95, 96], and 35 mM in
goats and cows [26, 29, 33]. Direct and indirect measurements
showed that milk glucose concentrations reflect intracellular
glucose concentrations of MECs; these concentrations are 0.1~
0.5 mM in rats, goats, and cows and ~1.3 mM in humans [40,
9799]. Thus, a steep glucose concentration gradient exists
across the basolateral membrane of MECs (Fig. 2).
Consistently, glucose transport into the MECs of guinea pigs,
rats, mice, and cows has been shown to be specific, saturable,
Na+-independent, specific to D-glucose, D-mannose, D-xylose,
and D-galactose, and inhibitable by cytochalasin-B or phloretin
[1116, 100], indicating a facilitated diffusion process. The K m
values for the uptake of 2-DG, 3-O-methyl-D-glucose (3-O-
MG, another non-metabolized glucose analogue), or glucose in
TJ
3-5 mM
0.1-0.3 mM
1 8
UDP-Gal
1
Basolaterial membrane
Apical membrane
8 SV
8
Interstitial space
SWEET
Capillary
8 12
1 When the glucose analogue 3-O-MG is infused into the udder
Glucose GLUT1 GLUT8 GLUT12 SGLT1
Fig. 2 Diagrammatic representation of a proposed model of glucose
transport in lactating bovine mammary alveolar epithelial cells. Glucose
is taken up from the interstitial fluid across the basolateral membrane by
GLUT1 and 8 and possibly SGLT1 and SWEETs. Glucose is transported
from the cytoplasm to the Golgi apparatus by GLUT1 and 8 and SGLT1.
The intracellular glucose equilibrates with the glucose in milk by GLUT8
and 12 and SGLT1. There may be GLUT trafficking between the plasma
membrane and intracellular pools for GLUT8 and 12 and between the
plasma membrane and the Golgi membrane for GLUT1. SV, secretory
vesicle; TJ, tight junction; and UDP-Gal, UDP-galactose
lactating human breast, bovine mammary epithelial cells and
rat mammary acini are 9.6, 8.29 and 16 mM, respectively [12,
14, 40], indicating that the affinity of the glucose transporter in
the mammary gland may be similar in different species. The
V max are 18.2 and 56 nmol/min per mg of protein in bovine
mammary epithelial cells and rat mammary acini, respectively
[12, 14], and were estimated to be 4.1 nmol/min per mg of
tissue in human breast [40].
However, mammary glucose transport also appears to have
a diffusion component and another carrier-mediated diffusion
component, which is not inhibited by cytochalasin-B or
phloretin. In lactating guinea pig mammary gland slices incu-
bated with 10 mM glucose, only approximately 50 % of the
glucose transport was inhibited by cytochalasin B [11], con-
sistent with the observation in lactating bovine MECs [14]. In
the presence of cytochalasin B, the glucose transport still
exhibited a saturable, carrier-mediated transport with a K m
of 1.25 mM [11]. In rat mammary acini, Threadgold et al. [12,
100] also observed a second non-specific transport process for
2-DG, which was also able to transport sorbitol, fructose, and
sucrose and was not inhibited by cytochalasin B or even D-
glucose. The nature of these cytochalasin B-insensitive com-
ponents is not clear. It would be interesting to investigate
whether the new family of SWEET proteins plays a role.
After entering MECs, glucose is phosphorylated by hexo-
kinases (HKs) to the phosphosugar G6P before being metab-
olized. Phosphorylation of glucose prevents its diffusion back
across the plasma membrane of cells and is thus critical for
retaining intracellular glucose. Thus, the rate of glucose phos-
0.1-0.3 mM phorylation should have a detrimental effect on the rate of
glucose transport across the basolateral membrane of MECs.
It has been shown that 3-bromopyruvate, an inhibitor of the
HK2 (the major hexokinase in MEC), significantly inhibits the
MILK
glucose uptake by bovine MECs [101]. HK activity may be
dependent on or control the rates of subsequent glucose me-
Lactose
tabolism [102]; high intracellular accumulation of G6P is
toxic to the cells because G6P strongly inhibits cell growth
12 and causes DNA damage [103, 104].
The rapid movement of glucose in both directions across the
apical membrane of MECs has also been observed and intracel-
lular glucose equilibrates quickly with the glucose in milk
(Fig. 2). When glucose is infused into the udder, 80 % of these
glucose molecules can be absorbed by the gland within 2 h [105].
SWEET
SWEET
with sorbitol, 3-O-MG is preferentially absorbed. In addition,
glucose and galactose are absorbed, but fructose is not [106].
These observations suggest that the apical membrane of MECs
also has a facilitative glucose transport system, whose transport
property is similar to that in the basolateral membrane. However,
because the glucose concentrations in milk and within cells are
much lower than the plasma concentration (see above), the
glucose transport activity in the apical membrane should be
marginal compared with that in the basolateral membrane.

Intracellular glucose and UDP-galactose must also cross the
Golgi membrane to reach the site of lactose synthesis. An early
study showed that lactose synthesis by Golgi-derived vesicles
could be inhibited by phloretin and cytochalasin B [11, 107],
indicating the presence of a GLUT-mediated facilitated transport
system in the Golgi membrane. However, the lactose synthesis
could also be inhibited by phlorizin, which is a Na+-dependent
glucose transport inhibitor. This finding suggests the co-existence
of a SGLT-mediated transporter system in the Golgi membrane.
The presence of carrier-mediated transport was further supported
by another study using an osmotic lysis method in which the
loaded [14C] lactose in the Golgi-membrane vesicles prepared
from lactating rat mammary gland could be partially lost after
exposure to glucose in a time-, temperature-, concentration-, and
structure-dependent manner [108]. However, L-glucose, which
cannot be transported by glucose transporters [109111], was as
effective as D-glucose in the study. It was later proposed that the
Golgi membrane may have protein-formed pores that facilitate
monosaccharide transport with limited specificity [112114].
The pore property of the Golgi membrane is strongly challenged
by the late discovery of the presence of GLUT1 in the Golgi
membrane (see below). Thus, the precise mechanism of glucose
transport across the Golgi membrane remains to be elucidated.
Glucose Transporters Expressed in the Mammary Gland
Zhao et al. [115] examined the expression of GLUT1-5 in
lactating bovine mammary gland by Northern blotting analysis
using human cDNA probes. The lactating mammary gland
expressed only a high abundance of GLUT1 and low levels of
GLUT3-5. No GLUT2 messenger was detected. Because rela-
tively high abundances of GLUT2, 4 and 5 transcripts were
detected in the other examined bovine tissues, the low or no
expression of these transporters in the mammary gland was not
due to the possible limited cross-hybridization between bovine
RNA and human probes. The strong expression of the GLUT1
protein of 42 kDa, but not of GLUT4, was later confirmed in
both lactating and non-lactating bovine mammary glands [116,
117]. Interestingly, a different size of GLUT1 of ~38 kDa was
detected in the non-lactating gland [116]; this GLUT1 may
reflect different post-translational modifications of the protein at
this stage. After the new GLUTs were discovered, GLUT8 and
GLUT12 messengers were also detected in the lactating bovine
mammary gland at apparent medium or low levels [88, 118].
High levels of GLUT1 expression have also been demon-
strated in rat [119122], mouse [123], and human [124, 125]
mammary glands. Expression of the high capacity GLUT2
was not detected in rat mammary gland [119, 121], as in
bovine mammary gland, but was observed in human breast
tissue [126]. In rat mammary gland, GLUT4 was detected but
only in virgin rats and during early pregnancy [119, 120].
Expression of GLUT4 decreases progressively during
J Mammary Gland Biol Neoplasia
pregnancy and becomes undetectable during late pregnancy
and lactation. This change is attributed to the progressive
decrease in the percentage of adipocytes in the gland during
pregnancy [119, 120]. No GLUT3 and 5 were observed in
human and rat mammary glands [120, 126]. In addition,
expression of GLUT 8 and 12 was shown in rat mammary
gland [122, 127].
Surprisingly, Shennan and Beechey (1995) observed that
changes in the Na+ gradient across the basolateral plasma mem-
brane of MECs rapidly stimulates the rate of 3-O-MG efflux
from the cells in lactating rat mammary tissue explants [128],
indicating the presence of SGLT-mediated transport. However,
the efflux was shown to be insensitive to the SGLT inhibitor
phlorizin. Nevertheless, the messenger and/or protein of SGLT1
were detected in lactating and/or non-lactating bovine, rat and
human mammary glands [128131]. Only the expression of
SGLT1, but not GLUT1, was detected in epithelial cells isolated
from fresh human milk [131]. In addition, a low level of SGLT2
mRNA was detected in bovine mammary gland [132]. The
physiological roles of these SGLTs in the mammary gland re-
main to be studied. SGLTs also serve as water and urea channels
and also transport anions, vitamins, and short chain fatty acids,
therefore, these functions in the mammary gland should also be
considered.
The mouse mammary gland also expresses SWEET1, whose
expression is induced during lactation [92]. These observations
imply a role of SWEET1 in milk synthesis. Because of the Golgi
localization of HsSWEET1, Chen et al. [92] proposed that
SWEET1 is possibly involved in glucose uptake by the Golgi
for lactose synthesis. However, this proposal is not supported by
the following observations: 1) the glucose transport in the Golgi
membrane is sensitive to the GLUT inhibitor phloretin [107],
whereas SWEET1 activity is insensitive to another GLUT inhib-
itor, cytochalasin B [92]; and 2) glucose transport in the Golgi
membrane has a low substrate specificity to monosaccharides
with no transport activity for disaccharides [108], whereas
SWEET1 does not appear to transport mannose, fructose, or
galactose but may transport sucrose [92]. Nevertheless, collabo-
rative functions of SWEET1 with GLUT1 in the Golgi mem-
brane cannot be excluded.
Cellular and Subcellular Localizations of Glucose
Transporters in the Mammary Gland and Their
Physiological Functions
Cellular and subcellular localizations of glucose transporters
determine their physiological functions. In the mammary
gland, milk synthesis occurs in MECs, in which the
basolateral, apical, or Golgi membrane localization of a trans-
porter may indicate its function in glucose uptake from the
blood stream, glucose secretion from the cell to milk, and
Golgi glucose uptake, respectively.
J Mammary Gland Biol Neoplasia
Strong expression of GLUT1 has been observed in the
MECs of bovine [116, 117], rat, [120, 122] and mouse [123]
mammary glands by immunochemistry and immunofluores-
cence staining. In lactating rat MECs, GLUT1 is mainly
present on the basolateral membrane with a barely detectable
presence on the apical membrane, as shown by immunofluo-
rescence and immunoelectron microscopy [120, 122, 127].
However, in bovine mammary gland, clear GLUT1 staining
was also observed on the apical membrane of MECs at 1 day
before parturition and during lactation, in addition to strong
staining on the basolateral side [117].
The predominant presence of GLUT1 in the basolateral
membrane of lactating MECs is consistent with the glucose
uptake kinetics in mammary tissue and MECs. Although the
plasma glucose concentration in cows is significantly lower
than that in humans, bovine and human GLUT1 exhibit a
similar transport Km , ~1012 mM, to 2-DG when expressed
in Xenopus oocytes [110]. This Km matches well with the
transport K m (816 mM) detected in lactating human breast,
bovine mammary epithelial cells, and rat mammary acini (see
above). In addition, the substrate specificity of GLUT1 (glu-
cose > mannose and galactose and no transport for fructose)
[110] is consistent with the transport specificity observed in
MECs [100].
In addition to be detected in plasma membrane-enriched
membrane fraction of lactating rat MECs, GLUT1 was also
detected in Golgi-enriched membranes [121]. Quantitative
Western blotting analysis showed that GLUT1 represents the
major glucose transporter species in plasma membranes and
about half of the glucose transporters in Golgi membranes.
Presence of GLUT1 in the Golgi membrane of MECs was also
observed in mice during late pregnancy and lactation but not
during the virgin and involution stages [123].
In rat mammary gland, GLUT8 is expressed in both MECs
and endothelium but absent from myoepithelium [127]. In
MECs, GLUT8 appears to be dispersed within the cells.
However, fairly strong GLUT8 staining was observed at both
the apical and basolateral membrane of lactating bovine
MECs (Finucane and Zhao, unpublished preliminary obser-
vations). GLUT12 was observed in the cytoplasm of MECs at
day 20 of pregnancy and at postpartum days 1 and 6 and on
the apical membrane during lactation [122].
Based on the above observations, I propose a general
model of glucose transport processes in MECs, shown in
Fig. 2. Glucose is taken up across the basolateral membrane
predominantly by GLUT1 with possible participations by
GLUT8, SGLT1, and SWEET. Glucose is taken up by the
Golgi apparatus through the collaborative functions of
GLUT1 and SGLT1. Glucose is released from the cytoplasm
to the milk across the apical membrane by GLUT8, GLUT12,
and SGLT1 in cows, mice, and rats, but most likely mainly by
SGLT1 in humans because there is a relatively high glucose
concentration in human milk [40] (there is a strong outward
Na+ concentration gradient across the apical membrane of
MECs [133]).
Glucose transport across the endothelial cells of capillary
vessels in the mammary gland remains poorly understood but
may likely be mediated mainly by GLUT1 because of its major
role in transporting glucose across blood-tissue barriers [71, 72].
Regulation of Glucose Transport in the Mammary Gland
Regulation of glucose homeostasis is a fundamental regulatory
function of all life, from bacteria to mammals [52, 134, 135].
However, the provision of glucose to the mammary gland for
milk synthesis is a metabolic priority in lactating mammals that
allows the species to survive, a condition referred to as
homeorhesis [136]. This priority is maintained by coordinated
homeorhetic regulation in body tissues even at the cost of break-
down of body homeostasis. Regulation of glucose transport in
the mammary gland plays a key role in meeting this priority.
Developmental Regulation
The mammary glucose transport rate increases approximately
40-fold from the virgin to the mid-lactation stage in rodents
and sharply declines during involution [137, 138]. These
changes are associated with marked changes in V max , but not
K m , suggesting that the number of transporters, rather than the
affinity of the transporter, is developmentally regulated. The
GLUT1 and GLUT8 expression in the mammary gland shows
consistent progressive increases from pregnancy to early lac-
tation in mice [88, 123], rats [119, 120], and cows [139, 140].
In the bovine mammary gland, the GLUT1 mRNA level
increases hundreds-fold from 40 days prepartum to 7 days
postpartum (Fig. 3), and the expression of GLUT8 and 12 as
well as that of SGLT1 and 2 is increased 4- to 10-fold (Fig. 3)
[139]. These increases suggest that all these transporters play a
role in lactation. GLUT1 mRNA is decreased after early lac-
tation, following the lactation curve [140]. The GLUT1 protein
level is lower in the bovine mammary gland at lactation day
181 compared with that at lactation day 118 [116]. The mam-
mary expression of GLUT1 mRNA and protein markedly
declines during abrupt weaning in mice and rats [120, 123]
and during the dry stage in cows [141]; this effect is not due to
the decrease in plasma prolactin concentration [120].
At least some of these developmental changes in GLUT
expression in the mammary gland result from the changes in
different cell populations during these stages. Unlike other
tissues, the mammary gland undergoes major development
after birth, especially during pregnancy, involving high rates
of growth and functional differentiation [142, 143]. Initially,
the epithelium invades the stroma (e.g., adipose and connec-
tive tissues) during tubule formation and the branching

Fig. 3 Developmental changes of GLUT1, GLUT8, GLUT12, SGLT1,
and SGLT2 mRNA expression in the bovine mammary gland at 40,
20, 7, and 7 days relative to parturition. Data were analyzed by
quantitative reverse transcription PCR from the mammary tissues taken
by biopsy from 12 Holstein cows at each stage. The mRNA levels of each
transporter were normalized to -actin levels. The figure is adapted with
permission from Zhao and Keating (2007) [139]
processes to establish a network of mammary ducts. During
pregnancy, the alveolar epithelium is developed to form milk
secretion units of alveoli. These epithelial cells are functionally
differentiated to produce milk during late pregnancy. The devel-
opment of mammary ducts and alveoli occurs well into the
lactation stage in many species. Thus, the populations of different
cell types, particularly the percentages of the epithelial cell and
adipose cell populations, change dramatically during mammary
development stages from virgin to lactation. These changes
undoubtedly affect the gene expression profiles in mammary
tissue. The decrease in GLUT4 expression in the mammary
gland from the virgin stage to late pregnancy as a result of the
decreasing adipose cell population is a good example [120]. The
concomitant increase in GLUT1 expression is at least partially
due to the increase in the epithelial population because epithelial
cells express much higher levels of GLUT1 than do adipocytes
[115]. However, the relatively small changes in epithelial cell
population cannot account for the dramatic increase in GLUT1
expression during the transition period from late pregnancy to
early lactation, which must mainly result from the enhanced
GLUT1 expression in these cells. It is not clear how much
changes in the expression of other transporters during mammary
development contribute to the changes in cell types because the
expression of these transporters in these cells is unclear.
Fasting
In lactating rats, the rate of glucose uptake by the mammary
gland decreases by 90 % due to overnight starvation [138]. In
lactating mice, the carrier-mediated uptake of 3-O-MG in
J Mammary Gland Biol Neoplasia
MECs reduces by 70 % after fasting for 16 h coupled with a
sharp decrease in the V max of the transport activity and the
number of cytochalasin-B-binding sites in the plasma membrane
of the cells [13]. However, the transport activity in fasted animals
can be quickly recovered by re-feeding [13, 138, 144].
Endocrine Regulation
The progressive increases in the glucose transport rate and the
expression of glucose transporters in the mammary gland
during pregnancy and early lactation imply that mammary
glucose transport may be regulated by lactogenic hormones.
In support of this notion, prolactin was found to stimulate 2-
DG uptake in mammary explants [145]. Disruption of prolac-
tin signaling in mice by a knockout of galanin or a single
prolactin receptor allele or by treatment of animals with a
phosphor-prolactin mimic all caused a failure of secretary
activation coupled with a decrease in GLUT1 expression in
the mammary gland [146]. Inhibition of prolactin secretion by
bromocriptine in lactating rats and mice also caused a decrease
in mammary GLUT1 expression [147, 148]. Furthermore, the
lactogenic hormone complex (prolactin and glucocorticoids)
induced a 15-fold increase in GLUT1 protein in CIT3 mouse
MECs [149]. In contrast to these previous observations, how-
ever, the lactogenic hormone complex failed to stimulate the
expression of GLUT1, 8, and 12 in bovine mammary ex-
plants, primary bovine MECs, and HC11 mouse MECs, al-
though the complex dramatically stimulated the expression of
milk proteins and lipogenic genes in our recent study [150].
The reasons for the discrepancy between our results in dairy
cattle and those of previous studies in rodents are not clear.
The difference may reflect the species differences between
cattle and rodents, but the expression of GLUTs was also not
affected by the lactogenic hormones in the HC11 mouse
MECs in our study. In addition, in support of our observations,
bromocriptine treatment of lactating rats for 24 h had no effect
on the GLUT1 protein levels in the mammary gland, although
the treatment decreased the GLUT1 mRNA levels [120].
Despite a 15-fold increase in GLUT1 protein in CIT3 cells
treated by the lactogenic hormone complex, the glucose up-
take by these cells was surprisingly decreased [149]. Thus, the
regulation of glucose transport by lactogenic hormones in the
mammary gland requires further investigations.
The lack of expression of GLUT4 in MECs [116, 119121]
is consistent with many observations that glucose uptake and
glucose transport in the mammary gland during lactation is
insensitive to insulin regulation (see above).
Injection of exogenous growth hormone increases glucose
uptake by the mammary gland without affecting the plasma
glucose concentration in dairy cows [36] and the glucose
concentration in milk in dairy goats [37]. These observations
suggest that growth hormone may stimulate glucose transport
J Mammary Gland Biol Neoplasia
in the mammary gland. However, the treatment of lactating
cows with exogenous growth hormone for 63 days did not
change the expression of GLUT1 mRNA and protein in the
mammary gland [151]. Thus, increased mammary glucose
uptake by exogenous growth hormone may mainly result from
increased blood flow to the mammary gland [152], rather than
from inducing GLUT expression in MECs.
Hypoxia Regulation
The insensitivity of GLUT expression to lactogenic hormones
in mammary tissue and MECs in our recent study [150] led us
to investigate a new hypothesis that the enhanced GLUT
expression and glucose uptake in the mammary gland from
mid-pregnancy to early lactation is stimulated by a local
chronic hypoxia [153]. Hypoxia is a physiological or patho-
logical condition of a deprivation of an adequate oxygen
supply in the body as a whole or within a tissue. During
hypoxia, many tissues and cells undergo a series of physio-
logical changes, including increased glucose uptake and en-
hanced anaerobic metabolism, to defend themselves against
the low oxygen supply [154156]. These effects are mainly
mediated by the heterodimeric transcription factor hypoxia
inducible factor 1 (HIF-1), which consists of an subunit
and a subunit. HIF-1 is constantly expressed in cells,
whereas HIF-1 is under tight regulation by the oxygen levels
within the cells. Under normoxia, HIF-1 is hydroxylated
[157], leading to its degradation by the proteasome
[158160]. Hypoxia can stabilize HIF-1 and the HIF com-
plex, resulting in HIF-1 translocation into the nucleus to
initiate expression of target genes, such as GLUT1 [154156].
Our new hypothesis is mainly based on two lines of evi-
dence. 1) From pregnancy to lactation, the metabolic rate of
the mammary gland increases to support mammary growth,
lactogenesis, and lactation. The mammary O2 uptake is steadi-
ly increased during late pregnancy and reaches its highest
levels during early lactation [30]. It is likely that this increas-
ing oxygen consumption results in a localized chronic hypox-
ia. 2) Mice with a HIF-1 deletion selective to the mammary
gland have impaired mammary differentiation and lipid secre-
tion [161]. These animals exhibit lower milk yield and striking
changes in milk composition. In addition, in these animals,
GLUT1 expression decreases by 60 % by day 16 of pregnan-
cy. These observations support a role of hypoxia in mammary
development, GLUT1 expression, and lactation.
Recently, to test our new hypothesis, we first examined the
hypoxia conditions in the mouse mammary gland from the virgin
state to the lactation state using the hypoxia marker pimonidazole
HCl (http://www.hypoxyprobe.com/). Pimonidazole HCl is a
chemical that forms adducts with thiol groups in proteins,
peptides, and amino acids in hypoxic cells. This binding can be
detected by immunohistochemical staining using specific
antibodies to pimonidazole adducts. By injecting mice with
pimonidazole HCl and by the subsequent immunoperoxidase
staining of the mammary tissues, we observed a hypoxic
mammary gland in all examined stages, but the staining was
stronger during late pregnancy and early lactation (Shao and
Zhao, unpublished preliminary observations). We then studied
the effects of hypoxia on GLUT expression in bovine MECs.
Hypoxia (below 5 % O2) markedly stimulated glucose uptake
and the expression of GLUT1 mRNA and protein in bovine
MECs. In contrast, hypoxia decreased the GLUT8 mRNA
expression in these cells. Robust induction of HIF-1 protein
was observed in bovine MECs, and an siRNA against HIF-1
completely abolished the up-regulation of GLUT1 by hypoxia
but had no effect on GLUT8 expression (Shao and Zhao, un-
published preliminary observations). These results support the
proposal that hypoxia may be responsible for the dramatic in-
creases in glucose uptake and GLUT1 expression in the mam-
mary gland during the transition period from mid-late pregnancy
to early lactation. Increased glucose uptake in hypoxic cells even
with decreased expression of GLUT8 demonstrated the predom-
inant role of GLUT1 in the glucose uptake of MECs. It is also
likely that hypoxia causes metabolic changes in MECs, such as
stimulating anaerobic metabolism as in cancer cells [162].
Other Regulations
The glucose transport rate in the cells can be regulated by
mechanisms other than changes in GLUT expression. The
regulations of the subcellular distribution and transport kinet-
ics of glucose transporters are two important examples.
The regulation of the subcellular localization of glucose
transporters in MECs is indicated by the following studies. 1)
The sharp decrease in the V max of the transport activity and the
number of cytochalasin-B-binding sites in the plasma mem-
brane of MECs in fasted mice can be completely reversed by
refeeding for only 23 h, suggesting a redistribution of the
glucose transporter to the plasma membrane [13, 138]. 2) In
CIT3 mouse MECs, most GLUT1 is targeted to the plasma
membrane in basal growth medium, but upon stimulation by
prolactin and hydrocortisone, translocation of GLUT1 to an
intracellular brefeldin A-sensitive compartment was observed
[149, 163]. In addition, GLUT1 is retargeted from the Golgi to
the plasma membrane of MECs of mice during forced
weaning [123]. These observations indicate that the lactogenic
hormone complex may induce GLUT1 translocation from the
plasma membrane to the Golgi, which is consistent with the
observation that GLUT1 is only targeted to the Golgi of
MECs during late pregnancy and lactation [123].
Glucose transporters can be glycosylated [88, 118]. The N-
and O -glycosylation of GLUT1 have been observed to have
an important impact on its transport activity [164166]. In
CIT3 mouse MECs, the lactogenic hormone complex induces

GLUT1 glycosylation [149]. We have detected different sizes
of GLUT1 in lactating and non-lactating bovine mammary
glands [116], potentially representing a different glycosylation
status. Thus, the mammary gland may regulate its glucose
transport activity by changing the glycosylation of the
GLUTs.
However, despite the implications from the above obser-
vations, the subcellular translocation and post-translational
modifications of glucose transporters in MECs require further
investigations.
Glucose Transporters in Breast Cancer
It has been long known that most solid tumors, including
breast cancer, have high rates of glycolysis (Warburg effect)
and glucose uptake [167169]. An order of magnitude higher
uptake of 2-[18 F]-fluoro-2-DG (FDG) has been consistently
observed by positron emission tomography (PET) imaging
and magnetic-resonance spectroscopy in different clinical tu-
mors in vivo compared with that in the adjacent normal tissues
[170172]. The enhanced rates of glycolysis and glucose
uptake in tumors are mainly stimulated by hypoxia, which is
one of characteristic features of malignant solid cancers [167,
173, 174]. The rapidly growing tumors are exposed to hyp-
oxia as a consequence of an inadequate blood supply, which,
in turn, promotes tumor progression by providing an array of
effects on tumor cells that favor tumor survival under hypoxic
conditions [153], including up-regulation of the expression of
GLUT1 and 3 and several key glycolytic genes (such as
hexokinases 1 and 2, lactate dehydrogenase A, and pyruvate
dehydrogenase kinases 1 [162, 175, 176]) by HIF-1.
Positive GLUT1 staining was found in 3847 % of breast
carcinomas [124, 177, 178]. Expression of GLUT1 has been
found to be significantly correlated with a higher tumor grade
and progression [124, 125, 177, 178], larger tumor size [124],
and the estrogen receptor and progesterone receptor status [124,
177, 179]. High levels of expression of GLUT1 in tumors are
associated with poor survival [124, 177]. Consistently, overex-
pression or knockdown of GLUT1 expression in mouse mam-
mary tumor cells enhances or inhibits, respectively, their
growth in nude mice in vivo [180]. Thus, GLUT1 expression
is considered to be a marker of aggressiveness and of a worse
prognosis in breast carcinoma patients.
The absence of GLUT1 staining in some breast cancers
suggests that other glucose transporters mediate glucose uptake
in these tumors. Indeed, expression of GLUT3 and 12 have also
been detected in breast cancers. In a recent study by Krzeslak
et al. [181], the expression of GLUT3 protein was identified in
21 % of breast carcinomas, and the expression of GLUT3
mRNA was significantly associated with the tumor grade and
estrogen and progesterone receptor status. The expression of
GLUT12 was detected in 8 of 10 invasive breast tumors [90].
J Mammary Gland Biol Neoplasia
Conclusions and Prospective Applications
Mammary glucose uptake plays a critical role in milk produc-
tion because glucose is an essential and key substrate and fuel
for milk synthesis in MECs. Elevated glucose uptake is also
commonly observed in breast tumors and is strongly nega-
tively associated with patient outcome. Thus, understanding
the molecular mechanisms underlying glucose uptake in the
mammary gland is of fundamental importance. Our knowl-
edge in this area has been greatly advanced by the discoveries
of glucose transporters. It is now clear that multiple glucose
transporters are involved in this process, but large gaps still
exist regarding the physiological and pathophysiological role,
cellular and subcellular localization, and regulation (gene
expression, subcellular trafficking, and kinetics) of all indi-
vidual glucose transporters in mammary tissues. Further stud-
ies on these topics will help us to increase glucose utilization
by MECs for milk synthesis in dairy production and to inhibit
glucose utilization by cancer cells for breast cancer treatment
and prevention.
Acknowledgments I am indebted to the students, fellows, and collab-
orators for their contributions to the cited works of my laboratory at the
University of Vermont. The work of my laboratory has been supported by
various grants from the USDA, including the National Research Initiative
Competitive Grants no. 2005-35206-15267 and no. 2007-35206-18037
from the USDA National Institute of Food and Agriculture.
Disclosures No conflicts of interest, financial or otherwise, are declared
by the author.
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