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Secretion and Regulation of ApoB48 by Primary Hamster Intestinal Enterocytes

Comprehensive study on secretion and regulation of ApoB48 in primary hamster intestinal cells, revealing links to lipogenesis and MTP activity.

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Secretion and Regulation of ApoB48 by Primary Hamster Intestinal Enterocytes

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  1. 120 120 100 100 80 80 60 60 40 40 20 20 0 0 Secretion and Regulation of ApoB48 by Primary Hamster Intestinal Enterocytes Media A Cells Chase Time (min) 45 90 0 45 90 B C P < 0.05 P = 0.01 Control Control MG132 Oleate Labeled ApoB48 (% control) Labeled ApoB48 (% control) Media Total Cells Total Cells Media

  2. 100 90 80 70 60 50 40 30 20 10 0 Ex vivo evidence for oversecretion of intestinal apoB48 & Enhanced intracellular apoB48 stability in fructose-fed hamster enterocytes Media Cells D Fructose-Fed Chow-Fed Chase Time (Min) 0 45 90 45 90 E F Total apoB 48 Secreted apoB 48 100 90 80 Labeled ApoB 48( % of 0 Time) 70 p= 0.003 Labeled ApoB 48( % of 0 Time) p= 0.001 60 Chow-Fed 50 Fructose-Fed 40 30 20 10 0 0 20 40 60 80 0 20 40 60 80 100 100 Chase Time (Min) Chase Time (Min)

  3. In Vivo Production of TG & Intestinal ApoB48 in the Fasting State

  4. In Vivo Production of TG & Intestinal ApoB48 in the Postprandial State

  5. 180 160 140 120 120 100 100 80 80 60 60 40 40 20 20 0 0 Ex Vivo Evidence for Intestinal Overproduction of Lipoprotein Lipids and Increase MTP mass/activity p=0.04 p=0.0013 Labeled Cholesterol (% of Control) A C B p=0.003 800 Labeled Triglyceride (% of Control) Labeled Cholesteryl Ester (% of Control) p= 0.002 250 p=0.001 700 Chow-Fed 200 600 Fructose-Fed 500 p=0.0018 150 400 300 100 200 50 100 0 0 Media Media Media Cellular Cellular Cellular E Fructose-fed Chow-fed Chow-Fed Fructose-Fed  D  p=0.006   160 ApoB 48 Secretion (% of control) 140 120 100 MTP Protein Mass (% of Control) 80 60 40 20 0 0.5 1 1.5 2 2.5 3 3.5 0 MTP inhibitor (M) Chow-Fed Fructose-Fed

  6. 80 70 60 50 40 120 30 20 100 10 80 0 60 20 Density/Size Distribution of Intestinal Lipoproteins Evidence for Increased Number & Size A B Chow-fed p= 0.06 80 p<0.026 Fructose-Fed 70 60 50 p<0.024 % of Total Labeled APoB48 40 % of Total Labeled ApoB48 p<0.05 30 20 p<0.03 10 0 Large CM Small CM VLDL LDL HDL Particles< 1.006 Particles > 1.006 140 Fasting Postprandial C D p<0.01 120 p<0.0007 P=0.0001 P=0.008 100 80 Labeled ApoB48 (% of Fructose-fed) Labeled ApoB48 (% of Fructose-fed) 60 40 40 20 0 0 Total apoB48 CM apoB48 Total-apoB48 CM apoB48

  7. 110 100 90 110 80 100 70 90 60 80 50 70 40 60 30 50 20 40 10 30 0 20 10 0 350 300 250 200 150 100 50 0 Increased De Novo Lipogenesis in FF Enterocytes Evidence that ApoB48 Secretion is Linked to the Rate of De Novo Lipogenesis A p= 0.002 Fatty Acid Synthesis Labeled Fatty Acid (% as control) Labeled Lipid (% as control) Sensitivity of TG & FA Secretion to Cerulenin Cholesterol Fatty Acid Triglyceride 0 2 4 6 8 10 12 14 16 Chow-fed Fructose-fed Cerulenin g/ml C Labeled Secreted ApoB-48 (% as control) Sensitivity of ApoB48 Secretion to Cerulenin 0 2 4 6 8 10 12 14 16 Cerulenin g/ml

  8. 1000 800 600 400 200 0 Acute Fructose Feeding or Exposure Does NOT Affect Intestinal ApoB48 Secretion Two-day Fructose Feeding In Vitro Incubation of Enterocytes with Fructose 120 120 100 100 80 80 Labeled ApoB-48 (% of control) Labeled ApoB-48 (% of control) 60 60 40 40 20 20 0 0 Chow-fed Fructose-fed (2 days) Control Fructose (3mM ) 450 P < 0.01 P < 0.0001 400 350 [14C] Fructose Incorporation into TG & Cholesterol 300 Labeled cholesterol Labeled Triglyceride 250 200 150 100 50 0 hepatocytes enterocytes hepatocytes enterocytes

  9. p85 b b IR a a Mechanisms of Intestinal Lipoprotein Overproduction in Insulin Resistant/ Hyperinsulinemic States TG dietary monoglyceride Apical Surface Intestinal Enterocyte FA De Novo (FA) FABP TG Glucose + FABP-FA FA ER FAS & ACC TG TG SREBP1c MTP PL Golgi PKB (Akt) TG CE PI3-K SOS ApoB48 IRS Proteasome p110 Grb-2 Degradation SHP-2 NCK Basolateral Surface PTK PTK ApoB48 Lipoprotein Particles Insulin (??)

  10. Acknowledgements Collaborators:Gary Lewis- TorontoAndre Carpentier- SherbrookeSven Olof Olofsson - Sweden Janet Sparks - Rochester, NY Raphael Cheung – Windsor Michel Tremblay – Montreal Denny Trinh - Toronto Laboratory Group: Changiz Taghibiglou Mehran Haidari Steven Van Iderstine Wei QuiTaryne Chong Farhana Mahboob Biao ChenLeyla MangalogluLouisa Pontrelli Fariborz Rashid Rita Kohen Debbie Rudy 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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