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VLDL

Hepatic Synthesis and Secretion of VLDL. ApoB Gene Expression. Proteasome. ApoB mRNA Translation. 5'. 3'. ApoB mRNA. Degradation. Lipid Poor State. Membrane. ER. MTP. Lipid Rich State. VLDL Assembly. CE. C. PL. Lipid Poor State. VLDL. TG. Degradation. Hepatocyte. Secretion.

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VLDL

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  1. Hepatic Synthesis and Secretion of VLDL ApoB Gene Expression Proteasome ApoB mRNA Translation 5' 3' ApoB mRNA Degradation Lipid Poor State Membrane ER MTP Lipid Rich State VLDL Assembly CE C PL Lipid Poor State VLDL TG Degradation Hepatocyte Secretion Plasma VLDL

  2. Mechanisms of VLDL Overproduction in Insulin Resistance (Recent Progress) • Development of a Fructose-Fed Hamster Model of Insulin Resistance • Investigations into Mechanisms of Hepatic VLDL Overproduction • Investigations into Mechanisms of Intestinal Lipoprotein Overproduction • Assessment of the Efficacy of hypolipidemic agents and insulin sensitizers in ameliorating metabolic dyslipidemia

  3. Insulin Resistance Model Fructose-Fed Syrian Golden Hamster • Lipoprotein metabolism closely resembles that in humans • Hamster liver secretes VLDL containing only apoB100 with a density close to that of human VLDL • Hamsters develop hyperTG, hyperCHOL, & atherosclerosis in response to a modest increase in dietary cholesterol & saturated fat • Hamster can be madeObese, Hypertriglyceridemic,Hyperinsulinemic, andInsulin Resistantby carbohydrate feeding (particularly Fructose)

  4. Insulin Resistance Model Fructose-Fed Syrian Golden Hamster Male Syrian Golden Hamsters (80-100 grams) Plasma Glucose, TG, Chol, Insulin 60% Fructose Diet (2 weeks) Control Diet (2 weeks) Control Hamsters Fructose-fed Hamsters Plasma Analysis: Glucose, TG, Chol, Insulin Liver Perfusions >>>>>>Primary Hepatocytes Intestinal Fragments >>>>>>Primary Enterocytes Experiments on Hepatic & Intestinal Lipoproteins

  5. Insulin Resistance Model Fructose-Fed Syrian Golden Hamster Hypothesis I: Insulin Resistance Induces Hepatic VLDL Overproduction Published Data: Evidence for Development of Insulin Resistance: • Increased Plasma Insulin, FFA, Triglyceride • Reduced whole body insulin sensitivity (based on Euglycemic-Hyperinsulinemic Clamp Studies) Evidence for Development of Hepatic VLDL Overproduction: • Enhanced hepatic VLDL secretion In Vivo (Triton method) • Enhanced VLDL secretion by primary hamster hepatocytes ex vivo • Increased intracellular apoB stability • Enhanced MTP expression (mRNA, protein, activity) Adeli K. et al. (2000) J. Biol. Chem. 275: 8416-8425.

  6. Increased Plasma Triglyceride, FFA, & Insulin in Fructose-Fed Hamsters 5 A B p=0.0309 p= 0.0550 7.5 4 5.0 3 Plasma Triglyceride (mmol/L) Plasma Cholesterol (mmol/L) 2 2.5 1 Control Fructose-Fed 0 0.0 p=0.9452 C D E p=0.0045 p=0.0110 300 2 7.5 200 5.0 Free Fatty Acids (mmol/L) Plasma Glucose (mmol/L) Plasma Insulin (mmol/L) 1 100 2.5 0 0.0 0

  7. In Vivo Evidence of Insulin Resistance (Euglycemic-hyperinsulinemic Clamp) Reduced Insulin Sensitivity in Fructose-Fed Hamsters Control (n=10) Fructose fed (n=9) 6 3000 A B p < 0.01 p = ns 5 2500 4 2000 3 1500 Glucose (mmol/l) Insulin (pmol/l) 2 1000 1 500 0 0 60 6 p = 0.03 D C p < 0.01 ) 50 5 -1 ) .min -1 40 4 .min -1 30 3 mol.kg -1 .kg 2 l m 20 2 6 (10 Ginf ( 10 1 I S 0 0

  8. Enhanced Hepatic VLDL-apoB100 Secretion in Fructose-Fed Hamsters (In Vivo Triton WR 1339 Studies) 400 12 10 350 * 8 g/min) VLDL-apoB secretion 6 g/ml) 300 m ( 4 m 2 250 0 VLDL-apoB ( 200 Control 150 Fructose fed 100 20 40 60 80 0 100 Time (min)

  9. Overproduction of VLDL-apoB by Hepatocytes from Fructose-Fed Hamsters ApoB100 500 * 400 VLDL apoB Secreted (% of control) 300 200 100 0 Control Fructose-Fed

  10. 250 Protein Mass 200 P=0.011 150 MTP Protein Mass (percent of control) 100 50 0 Control Fructose-Fed ) 20 Lipid Transfer Activity mRNA 250 P<0.02 15 total RNA 200 P=0.042 150 10 MTP RNA MTP Activity (Percent of Control) g 100 m 5 / 50 pg 0 0 ( Control Fructose-Fed Control Fructose-Fed Evidence for Enhanced Hepatic Microsomal Triglyceride Transfer Protein (MTP) in Fructose-Fed Hamsters

  11. Insulin Resistance Model (Fructose-Fed Hamster) Hypothesis II: VLDL-apoB Overproduction is Linked to Hepatic Insulin Resistance Recent Data: Insulin Signaling Status in Hepatocytes: • Ex vivo Analysis of Insulin Receptor, IRS-1, PI3-kinase, PTP-1B in Control and Fructose-Fed Hamster Livers • In Vitro Analysis of Insulin Receptor, IRS-1, PI3-kinase, PTP-1B in Primary Hepatocytes Exposed to High Insulin Link between Insulin Signaling & VLDL-apoB Secretion: • In Vitro Analysis of ApoB Secretion in Primary Hepatocytes Exposed to High Insulin • Inhibition of Protein Phosphatases by Vanadate and its Impact on VLDL-apoB Secretion (J. Biol. Chem. (2002) 277, 793-803)

  12. Insulin Signaling Pathway Insulin  Plasma membrane Insulin Receptor Shp-2 Gab 1  IRS Proteins Y Y P P SHC Protein kinase CK2 P P CAP Grb2 p85 Grb2 PTP-1B crkII Cbl mSoS PI 3-Kinase RaS mSoS p110 Ser/Ther-p Vanadate Caveolae PTEN PDK1 (PDK2) RAF Akt MEK Glucose & Lipid metabolism p70rak ToR Gsk3 PDE BAD MAPK aPKCs 90rak Gene Expression/ mitogenesis Protein synthesis Glycogen synthesis Glucose transport Anti- lipolysis Anti- apoptosis

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