WT Il6-/-0.0

0.5

1.0

1.5

Panc

reas

Mas

s (g

) ShamPDAC

✱✱✱✱✱✱

Ad-lib FR80

100

120

140

160

180

Glu

cose

(mg/

dL) Sham

PDAC

✱

✱

Slc2a1

Slc2a3Hk2Ldha

Slc16a3

0

5

10

15

20 GlycolysisPair FedPDAC

*** *** *** ***

Impaired adaptation to negative energy balance in pancreatic cancer-associated wasting Heike Mendez BS1, Xinxia Zhu MD2, Brennan Olsen BS2, Daniel L. Marks MD, PhD2, Aaron J. Grossberg MD, PhD1,3,4

1Brenden Colson Center for Pancreatic Care, Oregon Health & Science University, 3181 SW Sam Jackson Park Road, Portland, OR, 97239, United States2 Department of Pediatrics, Oregon Health & Science University, 3181 SW Sam Jackson Park Road, Portland, OR, 97239, United States3 Department of Radiation Medicine, Oregon Health & Science University, 3181 SW Sam Jackson Park Road, Portland, OR, 97239, United States4 Cancer Early Detection Advanced Research Center, Oregon Health & Science University, 2720 SW Moody Ave, Portland, OR, 97201, United States

ObjectiveThe disease associated wasting condition, cachexia, is a

common complication of pancreatic ductal

adenocarcinoma (PDAC) that impacts quality of life and

portends poor survival. Undernutrition is a major

driver of wasting in PDAC, yet cachexia remains

refractory to nutritional supplementation. By

modifying nutritional challenges at different stages of

cachexia development, we sought to understand the

relative contributions of undernutrition and metabolic

reprogramming to adipose and skeletal muscle

wasting.

HypothesisPDAC impairs the adaptive response to metabolic

stressors, leaving the host vulnerable to wasting in the

context of negative energy balance

Methods• Adult � & � C57BL/6J or Il6-/- mice received orthotopic

PDAC tumor (from KrasG12D; p53R172H/+; Pdx1-cre mice) or

sham injec_ons.

• Mice were metabolically challenged by 50% food

restric_on (FR) beginning 3 days aber orthotopic tumor

injec_ons using 2x2 factorial study design

• Adipose and muscle mass were quan_fied using serial

whole animal NMR and raw inguinal fat pad and

gastrocnemius weight at _me of necropsy

• Blood glucose and ketones were measured using point-of-

care glucometer and ketometer, respec_vely

• Ketogenic poten_al was evaluated by fas_ng mice

overnight, followed by octanoate challenge (0.2 mg/kg)

• Liver metabolic gene expression measured using qPCR

• Food absorp_on (exocrine func_on) es_mated by

measuring fecal protease ac_vity

• Sta_s_cal Analysis

• NMR over _me – one way ANOVA

• Correla_ons between food intake and was_ng - Linear

regression

• Food intake and ketone release – repeated measures ANOVA

• 2x2 factorial comparisons – two way ANOVA

• Student’s t-test used to compare 2 groups

• *, p<.05; **, p<.01; ***, p<.001

Experiment Design

Results• Orthotopic PDAC tumors elicit progressive anorexia, fat wasting, and muscle

wasting over time.

• Loss of fat mass is closely correlated to food intake (r=0.6, p<.01), whereas

muscle loss was not (r=0.2, p=0.54)

• Fecal protease activity is unimpacted by orthotopic PDAC tumors

• Applying subchronic food restriction elicited equivalent adipose loss in both

PDAC and sham mice, but muscle loss uniquely in PDAC mice (FR x PDAC

pinteraction <.05).

• Because adaptation to metabolic stress is mediated largely by the liver, we

looked at macronutrient partitioning and hepatic metabolic gene expression.

• Serum glucose is reduced by FR and PDAC (p<.05 for each). Ketogenic

potential in fasting mice is reduced by PDAC (p<.05).

• Hepatic expression of glycolytic genes is increased by PDAC, whereas

gluconeogenic and ketogenic gene expression is reduced.

• Whole body knockout of Il6 does not impair growth of PDAC.

• Whereas Il6 knockout does not impact PDAC-associated fat loss, loss of IL-6

increases muscle mass and may ameliorate PDAC-associated muscle wasting

(Il6 x PDAC pinteraction =.06) and hypoglycemia (Il6 x PDAC pinteraction =.08)

Conclusions1. Fat loss in PDAC is a function of nutrition alone, whereas muscle loss

is a function of both undernutrition and increased metabolic susceptibility

2. Metabolic reprogramming evident early in PDAC growth

3. PDAC impairs normal hepatic adaptive responses to metabolic stress,

which may explain increased vulnerability to undernutrition

4. IL-6 may mediate some of PDAC’s metabolic effects on the liver

Figure 1. Fat loss, but not muscle loss, follows food intake in PDAC-bearing mice. Orthotopic PDAC

cachexia time course experiment. (A) experiment design. (B) H&E stain of pancreas tumor. (C) daily food intake. Changes in fat mass (D) and lean mass, (E) measured by NMR. Statistical comparisons

using one way ANOVA. *, p<.05; **, p<.01

Figure 2. PDAC bearing mice do not spare muscle during metabolic challenge. (A) Experiment design.

Mice terminated before changes in voluntary food intake. Inguinal fat pad (B) and gastrocnemius muscle (C) mass at termina_on. No changes in fecal protease ac_vity observed among groups (not

shown). Sta_s_cal comparisons using 2-way ANOVA. *, p<.05.

A. B. C.

Figure 3. PDAC alters macronutrient partitioning and metabolic gene expression in the liver. Blood

glucose (A) in ad-libitum fed and fasted PDAC-bearing mice. Blood ketone levels following octanoate challenge (B). Liver expression of genes involved in gluconeogenesis (C), glycolysis (D), and oxidation &

ketogenesis (E). Statistical comparisons using 2-way ANOVA (A, B) or t-test (C-E).

A. B.

Figure 4. IL-6 ablation ameliorates but does not ablate PDAC-associated muscle wasting and hypoglycemia. (A) Experiment design. Tumor growth was not different between WT and Il6-/- mice (B). The

decrease in food intake caused by PDAC was unaffected by Il6 ablation (not shown). Fat mass was

decreased in PDAC bearing mice, regardless of Il6 presence (C). Gastrocnemius mass (D) and blood glucose

(E) were decreased in PDAC bearing mice, but independently increased in Il6-/- mice. 2-way ANOVA (B-E).

A. B.

KPC PDAC cells

Inject cells

Day 0 8 14

Terminate

11

Shamday

7

day 10

day 14

-2

-1

0

1

2

3

NMR Δ Lean mass - pre-fast

Δ L

ean

mas

s (g

)

1 way ANOVA p=0.01

***

Shamday

7

day 10

day 14

-2.0

-1.5

-1.0

-0.5

0.0

0.5

NMR Δ Fat mass - pre-fast

Δ F

at m

ass

(g)

1 way ANOVA p=0.04

NMR ∆ Fat MassNMR ∆ Lean Mass

Daily Food Intake

WT (C57BL/6J)

1 2 3 4 5 6 7 8 9 10 11 12 13 140

1

2

3

4

5

Days after surgery

Dai

ly F

ood

Inta

ke (g

)

Daily Food Intake - 2 group

PDAC

Sham

1 2 3 4 5 6 7 8 9 10 11 12 13 140

1

2

3

4

5

Days after surgery

Dai

ly F

ood

Inta

ke (g

)

Daily Food Intake - 2 group

PDAC

Sham

A. B.

C. D. E.

NMR NMR NMR

KPC PDAC cells

Inject cellsFood restrict

Day 0 3 7

Terminate

G6pc Pc

kFbp1

0.0

0.5

1.0

1.5

2.0

2.5

Gluconeogenesis

*** *

mR

NA

exp

ress

ion

(fol

d ch

ange

)

Ad-lib FR0

2

4

6

8

10

Fat P

ad M

ass

(mg/

g in

itial

BW

)

Sham PDAC

✱

Ad-lib FR0

2

4

6

Gas

troc

mas

s (m

g/g

initi

al B

W)

Sham Tumor

✱

✱

interaction p<.05 *

WT (C57BL/6J)

0 45 90 135 1800.0

0.5

1.0

1.5

2.0

Ketones

Time (mins)

[Ket

ones

] (%

t 0)

PDAC

Sham

* * *

Octanoate (0.2 mg/kg)

Hmgcs2Ppara Cp

t1

Ppargc1a

0.0

0.5

1.0

1.5

2.0β-oxidation & ketogenesis

*** ** * **

C. D. E.

KetonesGlucose

WT Il6-/-0

2

4

6

Fat P

ad M

ass

(mg/

g in

itial

BW

) ShamPDAC

✱

ns

WT Il6-/-0

2

4

6

Gas

troc

mas

s (m

g/g

initi

al B

W)

ShamPDAC

✱

✱✱✱

KPC PDAC cells

WT vs Il6-/-

Inject cells

Day 0 10

Terminate

WT Il6-/-0

50

100

150

200

bloo

d gl

ucos

e (m

g/dl

)

ShamPDAC

✱✱

✱

WT Il6-/-0

2

4

6

Fat P

ad M

ass

(mg/

g in

itial

BW

) ShamPDAC

✱

ns

WT Il6-/-0

2

4

6

Fat P

ad M

ass

(mg/

g in

itial

BW

) ShamPDAC

✱

ns

WT Il6-/-0

2

4

6

Fat P

ad M

ass

(mg/

g in

itial

BW

) ShamPDAC

✱

ns

C. D. E.

WT Il6-/-0

2

4

6

Fat P

ad M

ass

(mg/

g in

itial

BW

) ShamPDAC

✱

ns

Ad lib