Glut1: establishing a new paradigm for HIV-1
infection by regulating glucose metabolism
and activation in CD4+ T cells in
HIV-1-positive subjects
1
Glucose metabolism in T cells
Glucose Transporter-1 (Glut1) belongs to
a family of glucose transporters
Fu et al, 2004; Barata et al, 2004; Maratou et al, 2007
Glucose
CD3 + 5ug/ml CD28
GLUT1
CD3 + 1ug/ml CD28
Glut1 and is the main glucose transporter
on T cells and is regulated by
immunological signals
Intracellular retention of glucose is
facilitated by hexokinase (Hx) activity
T cell
CD3
No stimulation
Hx
G-6-P
CD3 + 5ug/ml CD28
Glucose is metabolized via glycolysis or
oxidative phosphorylation depending on
the T cell activation status
Powel and Delgoffe, 2010; Finlay and Cantrell, 2011
CD3 + 1ug/ml CD28
Glycolysis
(Lactate + 2 ATP)
Oxidative
Phos.
(36 ATP)
Proliferation
Antiviral
response
Migration
Cell growth
Lipids
Aerobic
Oxidative
phosphorylation
Glycolysis
ATPs
236ATPs
Cell membrane
Viral
replication
Amino Acids
Protein
Resting
T cell
Activated
T cell
Nucleotides
DNA
Hypothesis: HIV-1 infection increases glucose
metabolism in T cells
Rate of Glycolytic Metabolism
Glucose
Glut1
HIV
Activated T cells
T cells
in HIV+
subjects
Glut1
T cells in HIV- subjects
Glut1
T cells in HIV+/cART subjects
Aim 1
Evaluate cell surface expression of glucose
transporter 1 (Glut1) on T cells from
HIV-, HIV+ and HIV+/cART subjects
Subject groups
HIV-
HIV+
HIV+/cART
Subjects (n)
25
45
35
Male (%)
96
97
100
-
79,1740 ±
78,094
<50
397 ± 193
496 ± 256
Viral load
(copies/ml)
CD4 cell
(cells/ul)
Method: Glut1 expression on T cells
Fresh blood
(The Alfred Hospital, Melbourne)
Lysed red blood cells
Surface markers: CD3, CD4, CD8
Metabolic marker: Glut1
Flow cytometry
Frozen PBMCs
(UWA, USA)
HIV-1 infection is associated with increased Glut1 on
CD4+T cells
Glut1 on CD4+ T cells
Glut1 on CD8+ T cells
p=0.0001
p<0.0001
% CD8+Glut1+
%CD8+Glut1+ T cellscells
p=0.0009
80
60
40
30
20
10
105
100
95
90
80
40
45
/c
A
R
IV
+
H
H
n= 25
T
IV
+
H
IV
+
H
/c
A
R
T
IV
+
H
IV
-
IV
-
0
0
H
% CD4+Glut1+
% CD4+Glut1+ T cells cells
100
35
n= 25
45
35
Inverse relationship between CD4+Glut1+ T cells
and absolute CD4 cell count in HIV+ subjects
% CD4 vs %CD4+Glut1+ cells
CD4 count vs %CD4+Glut1+ cells
B
% CD4+ cells
Absolute CD4 cell count
A
% CD4+Glut1+ cells
% CD4+Glut1+ cells
Increased glucose metabolic activity may
Annexin
andduring
7AAD+
(cell
contributeV+
to (apoptosis)
CD4+ T cell loss
HIV-1
death) cells haveinfection
high Glut1 expression
Aim 2
Glycolysis pathway
To access the functional
significance of Glut1
expression on CD4+ T
cells
CD4+ T cells in HIV+ subjects have increased
glucose uptake
Kinetics of glucose
uptake in total CD4+ T
cells
Glucose uptake
in total CD4+ T
p=0.03
cells
p=0.001
p= 0.04
60
40
20
40
20
CD4+Glut1-
n=4
SSC
80
CD4+Glut1+
60
(2-NBDG)
MFI
MFI 2-NBDG in CD4+ T cells
120
T
90
/c
A
R
60
IV
+
Time (min)
H
30
IV
-
0
IV
+
0
0
H
MFIMFI
of 2-NBDG
on CD4+ T cells
(2-NBDG)
100
Glut1+ T cells
take up more
glucose
H
2-NBDG
n=
8
8
8
The intracellular concentrations of glucose-6-phosphate and
HIV-1
infection increases aerobic glycolysis
L-lactate are elevated in CD4+ T cells from HIV+ subjects
in CD4+
T cells
vivo
Palmer
et al., AIDS
2012,inTUAA0104
Host metabolism and viral infection
Human cytomegalovirus requires a glycolytic
environment for replication
Yu et al., Trends Microbiol, 2011
Glut1-mediated metabolic pathway regulates HIV-1
infection in naïve CD4+
Loisel-Meyer et al., PNAS, 2012
Are CD4+Glut1+ T cells more permissible to HIV-1
infection?
CD4+Glut1+ T cells are more permissive to HIV-1
infection in vitro
PBMCs from study participants (n=4)
Time on cART: 6 ± 4.3 years
Plasma viral load: <50
CD4 T cell count: 274 ± 98
CD4+Glut1+ : 57.0 ± 18.9%
Incubate with HIV-1-GFP (72h)
(Absence of exogenous stimuli)
Glut1
OX40 (PI3K)
Flow cytometry
HIV-1 infection in CD4+Glut1+ T cell subsets
40
n= 4
% GFP+ cells
30
20
10
0
Glut1+ OX40+
Glut1+ OX40-
Glut1-OX40-
Glut1
Glut1
Glut1Glut1 expression
PI3K
activity and
PI3K
PI3K
are both required
for efficient
HIV-1
infection in vitro
PI3K
Summary
1. Circulating CD4+Glut1+ T are increased in HIVinfected subjects are not restored to normal levels
during cART and have high glycolytic activity,
2. The frequency of CD4+Glut1+ T cells correlates
inversely with absolute CD4 count and a proportion
of these cells are highly apoptotic
3. CD4+Glut1+ T cells with high PI3K activity are
more permissive to HIV-1 infection in vitro
Speculations: role of Glut1 in HIV-1 pathogenesis
CD4+T cell loss
HIV-1
CD4+Glut1+ T cells
(Targets)
Vicious cycle
PI3Kᵧ
mTORC1
Glut1
Glycolysis
HIV-1
Inhibition may limit glucose metabolic activity
and activation
Acknowledgement
Contact: cpalmer@burnet.edu.au
Centre for Virology
(Pathogenesis lab), Burnet Institute,
Melbourne, Australia
Mentor: Suzanne Crowe
Anthony Jaworowski
Jingling Zhou
Linda Lam
Volunteers
Maelenn Gouillou
Anna Hearps
Anna Maisa
Wan-Jung Cheng
Tom Angelovich
Centre for Infection and Inflammation Research,
School of Medical Sciences, UNSW
Andrew Lloyd- Amany Zekry Lab
David Simar
Benny Baharuddin
Division of Experimental Medicine at the
University of California, San Francisco (UCSF)
Mentor: Joseph Mike McCune
!!!!!
Centre for Virology
(Virology/latency lab) Burnet Institute,
Melbourne, Australia
Sharon Lewin
Suha Saleh
Gabriela Khoury
Candida da Fonseca Pereira
Department of Chemistry, University of the West
Indies, Kingston, Jamaica
Willem Mulder
Baker IDI, Melbourne, Australia
Darren Henstridge
Creative and Novel Ideas in HIV Research
(CNIHR) Administrative Team
Center for AIDS Research
Clinical Research Core
University of Washington, USA
Robin Mohr
Distribution of CD4+Glut1+ T cells
Ki67 and Glut1 expression
Glut1 and proliferation
Figure S4
A
B
C
Glucose
Oligomyci
n
2Deoxyglucos
e
Clinical characteristics of study groups (Cross-sectional)
CFAR, University of Washington, USA
Alfred Hospital/Burnet Institute, Australia
Variables
P value
Groups
n
HIV-1
(A)
HIV+
(B)
HIV/cART
(C)
A vs B
B vs C
A vs C
105
24/1
44/1
35/0
-
-
-
BMI (Kg/m )
2
82
23.0 ± 2.5
24.7 ± 5.1
24.6 ± 5.9
0.17
0.40
0.26
Age (years)
93
45.2 ± 20.5
44.0 ± 10.7
44.6 ± 11.5
0.31
0.46
0.28
-
11/25
-
-
-
-
-
CD4+ T cell count
(cells/ul)
63
-
397 ± 193
496 ± 256
-
0.06
-
% CD3+CD4+ T cells
105
54 ± 1
28 ± 14
38.7 ± 15
<0.0001
0.001
0.0001
Viral load (copies/ml)
72
-
79,174 ± 78,094
<50
-
-
-
Plasma Glucose
(mmol/L)
80
4.8 ± 0.4
5.1± 1.0
5.1 ± 0.9
0.11
0.41
0.13
Plasma Insulin
(μIU/ml)
48
9.2± 8.9
6.3 ± 7.6
14.5 ± 19.3
0.26
0.50
0.44
TNF (pg/ml)
48
6.0± 3.0
10.5 ± 6.0
10.6 ± 8.7
0.005
0.28
0.02
Sex (M/F)
MSM (self-reported)
22