0

0.5

1

1.5

2

2.5

Control Con + met T2Dx T2Dx +

Met

Con + Flu T2Dx + Flu Con + Met

+ Flu

T2Dx +

Met + Flu

GL

UT

4 C

ell

Su

rfa

ce

Pro

tein

(R

U)

Novel Mechanisms Regulating Glucose Transport in the Lung of Influenza-Infected and Non-infected Diabetic Mice

Stephanie S. Vivies1, Allison Campolo1,2, Samantha Lazarowicz1, Delanie Beevers1, Veronique A. Lacombe1,2

Department of Physiological Sciences, Center for Veterinary Health Sciences, Oklahoma State University, Stillwater, OK, 740781, Harold Hamm Diabetes Center, The University of Oklahoma2

Fig. 1: A) Prevalence of diabetes (top) and of death by pulmonary

infection (bottom). B) Regulation of glucose transport in the lung.

Diabetes will alter the regulation of glucose

transport in the lung, which will be rescued by

metabolic therapy.

The prevalence and morbidity due to diabetes has

consistently increased over the years, in addition to the

mortality caused by respiratory infections such as influenza

(Fig 1A).

Hyperglycemia has recently been identified as an

independent risk factor for pulmonary infections.

The mechanisms governing glucose transport in the lung,

especially in hyperglycemic patients, is not well understood.

Glucose is thought to diffuse passively from the blood to the

airway surface liquid, and glucose transporters (GLUTs)

may be required to actively transport glucose into the cell

from the airway (Fig 1B).

Infected intranasally with influenza

H1N1 (A/PR/8/34, 250 PFU)

1. Klekotka, RB., et al. (2015) The etiology of lower respiratory tract infections in people

with diabetes, Pneumonol Alergol Pol 2015;83(5):401-8.

2. Reading, PC, et. al (1998) Increased susceptibility of diabetic mice to influenza virus

infection: compromise of collectin-mediated host defense of the lung by glucose?

1998;72(8):6884-6887

3. Kohio, HP, et al. (2013) Glycolytic control of vacuolar-type ATPase activity: a mechanism

to regulate influenza viral infection. Virology 2013 Sep;444(1-2):301-9

Type 1 Diabetic Type 2 Diabetic

Non-infected

Upper lung collected

3 days post infection

Quantification of major GLUT

isoforms

(GLUT, -2, -4, -10, -12) in the

lung via Western blotting

A B

1. Control C57BL/6 mice

2. Control mice treated with

metformin (200 mg/kg/day)

3. Type 2 diabetic (T2Dx,

high-fat diet fed, 60% kcal

from fat) (Fig 3)

4. T2Dx mice treated with

metformin

1. Blood glucose

2. Body weight

In vivo: Ex vivo:

1. Control FVB/N mice

2. Type 1 Diabetic mice

(streptozotocin-induced,

T1Dx, 8 weeks)

3. Diabetic mice treated with

insulin (subcutaneous

pump, 8 weeks)

This study was funded by the Harold Hamm Diabetes Center, Oklahoma Center for

Respiratory and Infectious Disease (OCRID-CoBRE, NIH 1P20 GM103648) and

Oklahoma State University Center for Veterinary Health Sciences.

CENTER FOR VETERINARY HEALTH SCIENCES

15

17

19

21

23

25

27

29

31

33

Baseline Post STZ Post pump

week 1

Post pump

week 3

Post pump

week 5

Post pump

week 7

Control

T1Dx

T1Dx + insulin

0

100

200

300

400

500

600

Baseline Post STZ Post pump

week 1

Post pump

week 3

Post pump

week 5

Post pump

week 7

Blo

od

Glu

co

se (

mg

/dL

)

Control

T1Dx

T1Dx + Insulin

100

150

200

250

300

0 1 2 3 4

Control

T2Dx

Con + Metformin

T2Dx + Metformin

15

25

35

45

55

65

0 1 2 3 4

Control

T2Dx

Con + Metformin

T2Dx + metformin

*#

* #

*

#*#

*#*#

*#*#

*#*#

*#

Insulin Pumps

Inserted

Metformin

Treatment Started

Insulin Pumps

InsertedMetformin

Treatment Started

Months Months

A B A B

*#

Insulin Treatment Rescues Hyperglycemia in Type 1

Diabetic Mice

Bo

dy

Weig

ht

(g)

Fig. 4: Mean ± SE of [glucose] and body weight, n=8-10/group. A) Mice injected with

streptozotocin (STZ) became markedly hyperglycemic, while insulin-treated mice

maintained normoglycemia. B) Body weight was not different between groups.

*p<0.05 vs control, #p<0.05 vs baseline via 2-way RM ANOVA. T1Dx: Type 1 diabetic

mice.

Metformin Treatment Rescues Hyperglycemia in

Insulin-Resistant Diabetic Mice

Bo

dy

Weig

ht

(g)

Blo

od

Glu

co

se (

mg

/dL

)

Fig. 6: Mean ± SE of [glucose] and body weight, n=8-10/group. A) Mice fed a high-fat

diet became mildly hyperglycemic, while metformin-treated mice maintained

normoglycemia. B) High-fat diet mice, regardless of treatment, had significantly greater

body weight than either control group. * p<0.05 vs control, # p<0.05 vs baseline via 2-

way RM ANOVA. T2Dx: Type 2 diabetic mice.

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

Control T1Dx T1Dx + Insulin Control + Flu T1Dx Flu T1Dx + Insulin +

Flu

0

0.5

1

1.5

2

2.5

3

Control T1Dx T1Dx + Insulin Control + Flu T1Dx Flu T1Dx + Insulin +

Flu

Insulin Treatment Rescues Alterations of GLUT

Protein Expression in the Lung of Influenza-infected

and Non-infected Type 1 Diabetic Mice

GLUT4

55 kDa

Β-Actin

40 kDa

0

0.5

1

1.5

2

2.5

Control T1Dx T1Dx + Insulin Control + Flu T1Dx Flu T1Dx + Insulin

+ Flu

GL

UT

4 P

rote

in

Exp

ress

ion

(R

U)

GL

UT

10 P

rote

in

Exp

ress

ion

(R

U)

GLUT10

52 kDa

Β-Actin

40 kDa

Non-infectedGLUT12

67 kDa

Β-Actin

40 kDa

GL

UT

12 P

rote

in

Exp

ress

ion

(R

U)

Fig. 5: Top panels: representative western blots; Bottom panels: Mean ± SE total

protein expression of A) GLUT4 B) GLUT10, C) GLUT12 (n=3-5/group) in adult

rodent upper lung. * p<0.05 vs. Control, † p <0.05 vs. Control + Influenza (Flu);

GLUT: Glucose transporter; T1Dx: Type 1 diabetic animal. Method: Western blotting.

A

B

C

*

*

*

†

†

Metformin Treatment Rescues Alterations of GLUT

Protein Expression in the Lung of Influenza-infected

and Non-infected Type 2 Diabetic Mice

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

Control Control +

Metformin

T2Dx T2Dx +

Metformin

Control + Flu Control + Met

+ Flu

T2Dx + Flu T2Dx + Met +

Flu

A

*†*

GLUT4

55 kDa

Β-Actin

40 kDa

GL

UT

4 P

rote

in

Exp

ress

ion

(R

U)

0

1

2

3

4

5

6

7

8

9

10

Control Control +

Metformin

T2Dx T2Dx +

Metformin

Control + Flu Control + Met

+ Flu

T2Dx + Flu T2Dx + Met +

Flu

C

*

†*

GLUT12

67 kDa

Β-Actin

40 kDa

GL

UT

12 P

rote

in

Exp

ress

ion

(R

U)

Fig. 7: Top panels: representative western blots; Bottom panels: Mean ± SE total

protein expression of A) GLUT4 B) GLUT2, C) GLUT12 (n=3-5/group) in adult

rodent upper lung. * p<0.05 vs. Control, † p <0.05 vs. Control + Flu; 2-tailed t-test.

GLUT: Glucose transporter; T2Dx: Type 2 diabetic animal; Met: Metformin. Flue:

Influenza. Method: Western blotting.

Fig. 3: Control (left) and high-fat diet-

fed (right) mice. Obese mice were

hyperinsulinemic, insulin resistant,

and hyperglycemic.

Both insulin and metformin treatment rescued alterations of

GLUT-4, GLUT-10, and GLUT-12 protein expression in upper

lung of type 1 and type 2 diabetic mice, respectively.

In both T1Dx and T2Dx groups, the non-diabetic mice

demonstrated an upregulation of GLUT trafficking to the cell

surface when infected with the flu compared to the non-

infected mice.

These novel findings suggest that 1) the regulation of

glucose transport is altered in the upper lung during

hyperglycemia, potentially worsening the response to

influenza infection, and 2) in vivo insulin or long-term

metformin treatment rescues GLUT protein alterations in the

diabetic lung.

Insights gained from this study could lead to the identification

of novel metabolic therapeutic targets for diabetic patients

affected by concurrent respiratory infections.

Fig. 9: Schematic diagram of alterations in pulmonary glucose transport and utilization

during diabetes and how these alterations may cause increased viral replication2,3.

0

0.5

1

1.5

2

2.5

3

Control T1Dx T1Dx + Insulin Control + Flu T1Dx + Flu T1Dx + Insulin

+ Flu

Cel

l S

urf

ace

GL

UT

4

Pro

tein

Co

nte

nt

(RU

)

GLUT4

55 kDa

*

*

†

GLUT4

55 kDa

Con T1DxT1Dx

+ Ins Con T1Dx

T1Dx

+ Ins

Non-infected Infected

Con T2Dx

T2Dx

+ Met

Non-infected Infected

Con

+ Met Con T2DxT2Dx

+ Met

Con

+ Met

A

B

*

*

*†

Increased GLUT4 Trafficking to the Cell Surface in

the Lung During Influenza

Fig. 8: Top panels: representative western blots; Bottom panels: Mean ± SE cell surface GLUT4

protein expression in A) T1Dx and B) T2Dx (n=3-5/group) adult rodent upper lung. * p<0.05 vs.

Control, † p <0.05 vs. Control + Flu; 2-tailed t-test. GLUT: Glucose transporter; T2Dx: Type 2

diabetic animal; T1Dx: Type 1 diabetic animal; Ins: Insulin; Met: Metformin; Flue: Influenza.

Method: Photolabeling biotinylation assay.

Fig. 2: Schematic figure of the

biotinylated photolabeling assay,

enabling quantification of cell-surface

glucose transporters (GLUTs).

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

2

Control Control +

Metformin

T2Dx T2Dx +

Metformin

Control + Flu Control + Met

+ Flu

T2Dx + Flu T2Dx + Met +

Flu

BGLUT2

61 kDa

Β-Actin

40 kDa

GL

UT

2 P

rote

in

Exp

ress

ion

(R

U)