Outline Potential Biomarkers for Use in Intestinal Adaptation Kelly A. Tappenden, Ph.D., R.D. Professor of Nutrition and GI Physiology 1. Intestinal Adaptation potential regulators 2. Intestinal mucosal changes 3. Digestive and absorptive outcomes 4. Biomarkers in clinical decision-making Department of Food Science and Human Nutrition University of Illinois at Urbana-Champaign Urbana, Illinois Jejunum is primary site of digestion and absorption Intestinal architecture facilitates functional capacity Short-Bowel Syndrome Parenteral Nutrition prevents malnutrition reduces intestinal adaptation systemic vs luminal delivery lack of specific nutrients, local factors, hormones and peptides? Intact Gastrointestinal Tract Short-Bowel Syndrome
Short-Chain Fatty Acids Products of fiber fermentation Acetate, propionate, butyrate comprise 83% of SCFAs Found in a fairly constant molar ratio of 6:2:1 Enhance structure and function in adult rodent small intestine mucosal proliferation in colon, ileum and jejunum (Sakata, 1987; Kripke et al., 1989; Koruda et al., 199) transporter activity (Tappenden et al., 1997, 1998; Tappenden and McBurney, 1998) Hypothesis TPN supplemented with butyrate alone will rapidly increase indices of intestinal adaptation in the neonate and these adaptive responses will be associated with increased plasma GLP-2 concentration.
Neonatal Piglet Model Similar in nutritional requirements, gastrointestinal physiology and metabolism Clinically relevant shortbowel syndrome and response to TPN Similar body composition to preterm infant Rapid growth and development TPN Experimental Design n = 12 Jugular Catheterization 8% Jejunoileal Resection SCFA 48 hours old 9 mm Bu 6 mm Bu 4h 12h 24h 3d 7d Acute Chronic Regardless of time, ileal villus length increased by supplemented treatments Villus Length (μm) 1 8 6 4 2 Control SCFA 9Bu 6Bu Ileum: Control < SCFA, P <.1 Number of PCNA Stained Cells/Crypt Ileal epithelial proliferation increased by supplemented treatments at all time points 2 16 12 8 4 4h 12h 24h 3d 7d Acute Chronic Ileum: Control < SCFA Treatments at 4h, 12h, 24h, 3d and 7d, P <.1 Control SCFA 9Bu 6Bu 12 hour, 1 Bax:Bcl-W Protein Abundance Butyrate induces anti-apoptotic protein ratio in ileum 12 1 8 6 4 2 Bax (21 kda) Bcl-w (21 kda) Control SCFA 9Bu 6Bu Ileum: Control > SCFA Treatments, P =.1 3 d 7 d Δ Isc/g (1 mm Glysar) Δ Isc/g (1 mm Glysar) PepT1 transport in jejunum is affected differently by Bu at 3 vs. 7 d 16 14 12 1 8 6 4 2 4 4 3 3 2 2 1 1 TPN SCFA 9Bu 6Bu * TPN SCFA 9Bu 6Bu *
Butyrate induced intestinal adaptations Apoptosis Control signal? Villus Height Butyrate Proliferation Glucagon-Like Peptide-2 Intestinotrophic peptide secreted from enteroendocrine L-cells (Drucker et al., 1996) Enhances structural and functional adaptations in adult small intestine crypt-villus architecture (Drucker et al., 1996; Tsai et al., 1997) enzyme and transporter activity (Brubaker et al., 1997; Cheeseman, 1997; Kato et al., 1999) Prevents parenteral nutrition-induced hypoplasia in adult and neonatal small intestine (Chance et al., 1997; Burrin et al., 2; Peterson et al., 2) Gattex, a GLP-2 analog, in Phase III clinical trials with parenteral nutrient/fluid dependent subjects with SBS Supplemented treatments increase plasma GLP-2 concentration at all time points What about humans with intestinal failure? Plasma GLP-2 (pmol/l) 3 3 2 2 1 1 Control SCFA 9Bu 6Bu Control < SCFA Treatments, P =.7 Intact Gastrointestinal Tract Short-Bowel Syndrome Therapeutic Role for in SBS? Analytical Methods Subjects with Jejunostomy n = 1 Subjects with Colon-in- Continuity n =.3,.1 or.1 mg/kg/d D D21 wash out D42.1 mg/kg/d Open-label, multicenter, pilot study Stable adult SBS subjects, ranging from.8 to 2 yrs since resection Dependent upon parenteral nutrients or fluid Morphological Analysis Crypt-villus architecture Mitotic index Gross Mucosal Analysis Ion and nutrient transport Mucosal composition (DNA, RNA, protein) Cellular Analysis RNA/protein content Transporter expression (SGLT-1,,, Bo, )
Villus Height (µm) Crypt Depth (µm) 9 8 7 6 4 3 2 1 1 1 2 2 increases jejunal villus height and crypt depth P =.24 P =.18 P =.3 P =. P =.29 P =. D D21 D42 D D21 D42 1 2 3 4 6 Crypt Depth (µm) Number of Cells in Mitosis reversibly increases jejunal mitotic index 2. 2. 1. 1.. P =. P =.3 P =.33 Day Day 21 Day 42 Day Day 21 Day 42 1..8.6.4.2 Number of Cells in Mitosis DNA (μg RNA/mg mucosa) increases jejunal cellularity 1.6 P =.48 P =.29 1.2.8.4. Day Day 21 Day 42 Day Day 21 Day 42 RNA (μg RNA/mg mucosa) Day Day 21 Day 42 Subjects w/ jejunostomy 1.41 ±.32 2.99 ±.98* 1.84 ±.42 Subjects w/ colon 1.2 ±.26 1.6 ±.29 1.23 ±.44 Cellular RNA concentration (RNA/DNA) Subjects w/ jejunostomy 2.99 ±.7 3.32 ± 1.1 2.36 ±.67 Subjects w/ colon 2.27 ±.77 1.69 ±.61 1.88 ± 1.1 Protein (μg RNA/mg mucosa) Subjects w/ jejunostomy 42.6 ±.7 47. ± 6.7 44.8 ± 3.6 Subjects w/ colon 4.4 ± 3.2 41.6 ±. 1.4 ±.7 Cellular protein concentration (RNA/DNA) Subjects w/ jejunostomy 8. ± 14.9 64.3 ± 12.7 1.3 ± 8.2 Subjects w/ colon 7.9 ± 19.9 64.9 ± 14.6 87. ± 29.6 Mechanisms for teduglutide-induced structural adaptations Ion and Nutrient Transport Apoptosis? villus ht/ crypt depth cellularity mitotic index Short-circuit current ( ma) 1 9 8 7 6 4 3 2 1 Carbachol Serotonin Glutamine 1 2 3 4 6 7 8 Time (minutes) Following teduglutide
GLP-2 enhances jejunal active ion transport increases jejunal glucose transport Day Day 21 Day 42 Transmural Short-Circuit Current (μa/cm 2 ) Subjects w/ jejunostomy -11.4 ± 8. 4.9 ± 3.6* -. ± 3.6 Subjects w/ colon 27. ± 17.3 36.6 ± 3.2 6.44 ± 4.22 Transmural Resistence (Ω cm 2 ) Subjects w/ jejunostomy 24.8 ±.2 28. ± 7.6 31.6 ± 1.6 Subjects w/ colon 36. ±7.9 39.1 ± 9. 34.8 ± 7.3 Potential Difference (mv) Subjects w/ jejunostomy -.3 ±.9.3 ±.2.4 ±.7 Subjects w/ colon.78 ±.34.91 ±.61.26 ±.13 Δ in Isc (1 mm glucose) 9 8 7 6 4 3 2 1 P =.42 D D21 D42 9 8 7 6 4 3 2 1 D D21 D42 Δ in Isc (1 mm glucose) Key jejunal monosaccharide transporters increases jejunal, and protein mrna NC protein SGLT-1 H 2 O H 2 O H 2 O SGLT-1 H 2 O mrna NC protein NC mrna NC protein increases jejunal glutamine uptake Δ in Isc (1 mm glutamine) 3 P =.4 3 2 2 1 1 D D21 D42 4 4 3 3 2 2 1 1 D D21 D42 Δ in Isc (1 mm glutamine) increases nutrient uptake via multiple mechanisms SGLT-1 Bo SGLT-1 Brush Border Membrane Basolateral Membrane
increases jejunal serotonin-induced chloride secretion enhances intestinal structure and function 7 7 Δ in Isc (.1 mm serotonin) 6 4 3 2 1 Day Day 21 Day 42 Day Day 21 Day 42 6 4 3 2 1 Δ in Isc (.1 mm serotonin) villus height crypt depth cellularity Intestinal Epithelium H 2 O Jejunum > Colon Enterocyte SGLT-1 H 2 O Na + October 11, 27: Clinical Benefits Demonstrated with Low-Dose GATTEX in Phase 3 Short Bowel Syndrome Study. 83 patients with short bowel syndrome Design of multicenter prospective, randomized, double-blind, placebo-controlled study (n=16) placebo,. mg/kg/day or.1 mg/kg/day for 24 weeks primary endpoint: reduction of PN by 2% 46% of patients on low-dose responded (p=.7) 2% of patients on high-dose responded (p=.161) Subjects 83 PNdependent subjects with intestinal failure Optimize PN Intestinal Biopsy Stabilize PN -8 wks 4-8 wks. mg/kg/d (n=3).1 mg/kg/d (n=32) 24 wks Reduction in PN (L/week) Reduction in PN 1 4-1 -2.1 mg/kg/d -3-4 -. mg/kg/d -6 4. 4 8. 8 12. 16.1 6 2. 24. 28. 32. 36. 4. 44. 2. Week kg 62 61 6 9 8 Body Weight (Hydrational Status/Fluid Retention) p=.4 p=.18 D.23 D.73 D1.32 P=.77 Week 24/LD P=.11 Week 24/LD p=. Week 24/LD. mg/kg/d.1 mg/kg/d Jeppeson et al., UEGW 28
In a small patient subset, teduglutide led to numerical improvements in intestinal fluid absorption. Plasma Citrulline Levels Correlate with Functional Intestinal Mass Intestinal fluid absorption (g/72-hours) DW 8 DW 24 (end-of-study) Plasma citrulline levels: Normal plasma levels are 4 ± 1 µmol/l in adults Correlate with functional intestinal mass Correlate with the extent of villus atrophy and reduced functional mass Can differentiate between non-pn-dependent and PN-dependent SBS patients n=4. n=1.1 n=7 Total n=17 Crenn, Gastroenterol 2; Crenn, Gastroenterol 23 Plasma Citrulline (µmol/l) Increased Plasma Citrulline Levels in Treated Groups 4 3 3 2 2 1 1 Week 2 Week 24 Treated vs. placebo p<.1. mg/kg/d.1 mg/kg/d Messing et al., UEGW 28 Small intestinal villus height changed -19%, +4% and +39% in the placebo,. and.1 groups, respectively. Villus Height (µm) Crypt Depth (µm) 9 8 7 6 4 3 2 1 1 2 3 4 6..1 Total teduglutide teduglutide teduglutide Small Intestine End-of-study..1 Total teduglutide teduglutide teduglutide Large Intestine administration at. and.1 resulted in statistically significant villous height increases. Representative change in small intestinal mucosa following 24 weeks of. teduglutide administration. Change in Villous Height (µm) P =.2 P =.6 P =.649 +4% +39% -19%..1 End-of-study
Mucosal DNA, RNA and protein concentration not altered by teduglutide in the small intestine. Summary -induced expansion of the mucosal epithelium of adult patients with SBS and may therefore enhance their capacity to digest and absorb orally consumed nutrients...1..1..1 The DNA, RNA and protein composition of teduglutide remodeled mucosa did not differ from placebo. Take Home Message Strategies to enhance structural and functional characteristics of intestinal adaptation, such as short-chain fatty acids and GLP-2 therapy, promise to reduce parenteral nutrient requirements in individuals with intestinal failure, by targeted remodelling of the intestinal epithelium. Anne L. Bartholome, Ph.D., R.D. David M. Albin, Ph.D. Jens J. Holst, M.D., Ph.D. Michael I. McBurney, Ph.D. Alan B.R. Thomson, M.D., Ph.D. NIDDK R1 DK 7682 Acknowledgements