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Slides 29 38

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Slides 29 38

  1. 1. Secretion and Regulation of ApoB48 by Primary Hamster Intestinal EnterocytesIntestinal Enterocytes B C LabeledApoB48(%control) LabeledApoB48(%control) 0 20 40 60 80 100 120 Total Cells Media Control MG132 P < 0.05 0 20 40 60 80 100 120 Total Cells Media Control Oleate P = 0.01 Cells Media 0 45 90 45 90 A Chase Time (min)
  2. 2. Cells Media Fructose-Fed Chow-Fed Chase Time (Min) 0 45 90 45 90 D Chase Time (Min) Chase Time (Min) 100 100 0 10 20 30 40 50 60 70 80 90 0 20 40 60 80 Total apoB 48 LabeledApoB48(%of0Time) LabeledApoB48(%of0Time) p= 0.003 p= 0.001 Secreted apoB 48 0 10 20 30 40 50 60 70 80 90 100 0 20 40 60 80 100 Chow-Fed Fructose-Fed E F Ex vivo evidence for oversecretion of intestinal apoB48 & Enhanced intracellular apoB48 stability in fructose- fed hamster enterocytes
  3. 3. In Vivo Production of TG & Intestinal ApoB48 in the Fasting State Time (min) 55 60 65 70 75 80 85 Sf>400TGconcentration (mmol/l) 0 1 2 3 4 Sf >400 after Triton WR1339 Time (min) 55 60 65 70 75 80 85 Sf>400ApoB48concentration (µg/ml) 140 160 180 200 220 240 260 280 Time (min) 55 60 65 70 75 80 85 Sf100-400TGconcentration (mmol/l) 0 1 2 3 Sf 100-400 after Triton WR1339 Time (min) 55 60 65 70 75 80 85 Sf100-400ApoB48concentration (µg/ml) 90 100 110 120 130 140 150 160 170 Triglyceridesecretion 0.0 0.2 0.4 0.6 0 1 2 3 ApoB48secretion 0 4 8 12 16 Triglyceridesecretion 0.0 0.1 0.2 0.3 ApoB48secretion 0 2 4 6p = 0.002 p = 0.01 p = 0.14 p = 0.59 A B DC
  4. 4. In Vivo Production of TG & Intestinal ApoB48 in the Postprandial State Time (min) 55 60 65 70 75 80 85 Triglyceridelevel(mmol/l) 0.0 0.1 0.2 0.3 0.4 0.5 0.6 Time (min) 55 60 65 70 75 80 85 ApoB48level(µg/ml) 80 90 100 110 120 130 140 150 Time (min) 55 60 65 70 75 80 85 Triglyceridelevel(mmol/l) 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 Time (min) 55 60 65 70 75 80 85 ApoB48level(µg/ml) 50 60 70 80 90 100 Triglyceridesecretion(µmol/min) 0.00 0.01 0.02 0.03 0.04 0.05 0.06 0.07 Col 48: 0.07 Col 48: 0.06 Triglyceridesecretion(µmol/min) 0.00 0.02 0.04 0.06 0.08 0.10 Col 50: 0.08 Col 50: 0.09 0 1 2 3 ApoB48secretion(µg/min) 0 1 2 3 4 5 6 7 8 Col 52: 2.65 Col 52: 7.46 ApoB48secretion(µg/min) 0 1 2 3 4 5 6 7 8 Col 54: 3.85 Col 54: 7.26 Sf 100-400 after Triton WR1339 Sf >400 after Triton WR1339 A B C D p = 0.3 p = 0.003 p = 0.8 p = 0.007
  5. 5. 0 20 40 60 80 100 120 140 160 Fructose-Fed MTPProteinMass (%ofControl) Chow-Fed Fructose-Fed 0 20 40 60 80 100 120 0 0.5 1 1.5 2 2.5 3 3.5 MTP inhibitor (µM) ApoB48Secretion(%ofcontrol) Fructose-fed Chow-fed ∗ ∗ ∗ ∗ Chow-Fed p=0.006D E LabeledCholesterylEster (%ofControl) p= 0.002 0 50 100 150 200 250 Cellular p=0.001 Media B LabeledCholesterol (%ofControl) Chow-Fed Fructose-Fed 0 100 200 300 400 500 600 700 800 Cellular p=0.0018 p=0.0013A Media LabeledTriglyceride (%ofControl) Cellular 0 20 40 60 80 100 120 140 160 180 p=0.04 p=0.003 Media C Ex Vivo Evidence for Intestinal Overproduction of Lipoprotein Lipids and Increase MTP mass/activity
  6. 6. Fructose-Fed Chow-fed 0 10 20 30 40 50 60 70 80 Large CM Small CM VLDL LDL HDL p<0.05 p= 0.06 p<0.03 %ofTotalLabeledApoB48 A 0 20 40 60 80 100 120 140 CM apoB48 Total-apoB48 LabeledApoB48 (%ofFructose-fed) p<0.0007 p<0.01C 0 10 20 30 40 50 60 70 80 Particles< 1.006 Particles > 1.006 %ofTotalLabeledAPoB48 p<0.024 p<0.026 B 0 40 20 60 80 100 120 CM apoB48 Total apoB48 LabeledApoB48 (%ofFructose-fed) P=0.008 P=0.0001 DFasting Postprandial Density/Size Distribution of Intestinal Lipoproteins Evidence for Increased Number & Size
  7. 7. Increased De Novo Lipogenesis in FF Enterocytes Evidence that ApoB48 Secretion is Linked to the Rate of De Novo Lipogenesis 0 10 20 30 40 50 60 70 80 90 100 110 0 2 4 6 8 10 12 14 16 Cholesterol Fatty Acid Triglyceride LabeledLipid(%ascontrol) Cerulenin µg/ml 0 50 100 150 200 250 300 350 Chow-fed Fructose-fed LabeledFattyAcid(%ascontrol) 0 10 20 30 40 50 60 70 80 90 100 110 0 2 4 6 8 10 12 14 16 Cerulenin µg/ml LabeledSecretedApoB-48(%ascontrol) A C p= 0.002 Sensitivity of ApoB48 Secretion to Cerulenin Fatty Acid Synthesis Sensitivity of TG & FA Secretion to Cerulenin
  8. 8. 20 60 80 100 0 40 120 Chow-fed Fructose-fed (2 days) 0 20 40 60 80 100 120 Control Fructose (3mM ) 0 200 400 600 800 1000 enterocytes hepatocytes P < 0.01 LabeledApoB-48(%ofcontrol) LabeledApoB-48(%ofcontrol) Labeledcholesterol 0 50 100 150 200 250 300 350 400 450 enterocytes hepatocytes LabeledTriglyceride P < 0.0001 Acute Fructose Feeding or Exposure Does NOT Affect Intestinal ApoB48 Secretion [14 C] Fructose Incorporation into TG & Cholesterol Two-day Fructose Feeding In Vitro Incubation of Enterocytes with Fructose
  9. 9. Mechanisms of Intestinal Lipoprotein Overproduction in Insulin Resistant/ Hyperinsulinemic States Apical Surface Basolateral Surface FA monoglyceride FABP-FA FABP TG TG TG ApoB48 PL CE De Novo (FA) Insulin (??) TGdietary SOS Grb-2 IRS PI3-K p110 SHP-2 NCK PTKPTK β β α α IR PKB (Akt) SREBP1c FAS & ACC Glucose + FA MTP ERER GolgiGolgi Intestinal Enterocyte TG ApoB48 Lipoprotein Particles p85Proteasome Degradation
  10. 10. Acknowledgements Laboratory Group: Changiz Taghibiglou Mehran Haidari Steven Van Iderstine Wei Qui Taryne Chong Farhana Mahboob Biao Chen Leyla Mangaloglu Louisa Pontrelli Fariborz Rashid Rita Kohen Debbie Rudy Collaborators: Gary Lewis- Toronto Andre Carpentier- Sherbrooke Sven Olof Olofsson - Sweden Janet Sparks - Rochester, NY Raphael Cheung – Windsor Michel Tremblay – Montreal Denny Trinh - Toronto Funding: Heart & Stroke Foundation of Ontario Canadian Institutes of Health Research NSERC Hospital for Sick Children Merck-Frosst pfizer GlaxoSmithKline BBDC, University of Toronto

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