WT Il6 -/- 0.0 0.5 1.0 1.5 Pancreas Mass (g) Sham PDAC ✱✱✱ ✱✱✱ Ad-lib FR 80 100 120 140 160 180 Glucose (mg/dL) Sham PDAC ✱ ✱ Slc2a1 Slc2a3 Hk2 Ldha Slc16a3 0 5 10 15 20 Glycolysis *** *** * ** *** Impaired adaptation to negative energy balance in pancreatic cancer-associated wasting Heike Mendez BS 1 , Xinxia Zhu MD 2 , Brennan Olsen BS 2 , Daniel L. Marks MD, PhD 2 , Aaron J. Grossberg MD, PhD 1,3,4 1 Brenden Colson Center for Pancreatic Care, Oregon Health & Science University, 3181 SW Sam Jackson Park Road, Portland, OR, 97239, United States 2 Department of Pediatrics, Oregon Health & Science University, 3181 SW Sam Jackson Park Road, Portland, OR, 97239, United States 3 Department of Radiation Medicine, Oregon Health & Science University, 3181 SW Sam Jackson Park Road, Portland, OR, 97239, United States 4 Cancer Early Detection Advanced Research Center, Oregon Health & Science University, 2720 SW Moody Ave, Portland, OR, 97201, United States Objective The 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. Hypothesis PDAC 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 Kras G12D ; p53 R172H/+ ; 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 p interaction <.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 p interaction =.06) and hypoglycemia (Il6 x PDAC p interaction =.08) Conclusions 1. 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 Sham day 7 day 10 day 14 -2 -1 0 1 2 3 Δ Lean mass (g) ** * Sham day 7 day 10 day 14 -2.0 -1.5 -1.0 -0.5 0.0 0.5 Δ Fat mass (g) NMR ∆ Fat Mass NMR ∆ Lean Mass Daily Food Intake WT (C57BL/6J) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 0 1 2 3 4 5 Days after surgery Daily Food Intake (g) PDAC Sham A. B. C. D. E. NMR NMR NMR KPC PDAC cells Inject cells Food restrict Day 0 3 7 Terminate G6pc Pck Fbp1 0.0 0.5 1.0 1.5 2.0 2.5 Gluconeogenesis ** * * mRNA expression (fold change) Ad-lib FR 0 2 4 6 8 10 Fat Pad Mass (mg/g initial BW) ✱ Ad-lib FR 0 2 4 6 Gastroc mass (mg/g initial BW) ✱ interaction p<.05 * WT (C57BL/6J) 0 45 90 135 180 0.0 0.5 1.0 1.5 2.0 Time (mins) [Ketones] (% t 0 ) PDAC Sham * * * Octanoate (0.2 mg/kg) Hmgcs2 Ppara Cpt1 Ppargc1a 0.0 0.5 1.0 1.5 2.0 β-oxidation & ketogenesis *** ** * ** C. D. E. Ketones Glucose WT Il6 -/- 0 2 4 6 Fat Pad Mass (mg/g initial BW) ns WT Il6 -/- 0 2 4 6 Gastroc mass (mg/g initial BW) ✱✱✱ KPC PDAC cells WT vs Il6 -/- Inject cells Day 0 10 Terminate WT Il6 -/- 0 50 100 150 200 blood glucose (mg/dl) ✱✱ Sham PDAC ✱ Sham PDAC ✱ Sham PDAC ✱ C. D. E. Sham PDAC ✱ Ad lib