Role of Liver In Triglyceride Homeostasis. Larry L. Swift, Ph.D. Department of Pathology Vanderbilt University School of Medicine

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1 Role of Liver In Triglyceride Homeostasis Larry L. Swift, Ph.D. Department of Pathology Vanderbilt University School of Medicine

2 Metabolic Cycle of Fatty Acids and Triglycerides VLDLTG

3 Fatty Acids Aliphatic carboxylic acids usually having an even number of carbon atoms Chains may be saturated or unsaturated Position of double bonds described in relation to carboxy-terminus ( ) or methyl carbon ( or n)

4 Common Fatty Acids Fatty Acid C:DB, position Structure Stearic 18:0 Oleic 18:1 ω9 Linoleic 18:2 ω6 α-linolenic 18:3 ω3 γ-linolenic Arachidonic 18:3 ω6 20:4 ω6 Eicosapentaenoic 20:5 ω3

5 Fatty Acid Biosynthesis Takes place in the cytosol Acetyl CoA is substrate; palmitate (C16:0) is product Fatty acids are synthesized by a multi-enzyme complex, fatty acid synthase (FAS) Elongation beyond C16:0 and desaturation are catalyzed by enzymes associated with the endoplasmic reticulum

6 Amino Acids Fatty acids Pyruvate Glucose Pyruvate Denhydrgenase Cytosol Acetyl CoA Fatty Acids Mitochondria

7 Fatty Acid Biosynthesis First committed step of fatty acid synthesis pathway Acetyl CoA Carboxylase (ACC) is major regulatory site of fatty acid synthesis

8 Fatty Acid Biosynthesis Final product is palmitic acid, C16:0

9 Fatty Acid Synthase

10 Fatty Acid Elongation / Desaturation Mammals and plants Palmitate (16:0) elongation desaturation Stearate (18:0) Oleate 18:1 ( 9 or 9) Palmitoleate 16:1 ( 9 or 7) Longer saturated fatty acids * Plants only desaturation * -linoleate 18:3 ( 9,12,15 or 3) Other PUFAs * Linoleate 18:2 ( 9,12 or 6) -linoleate 18:3 ( 6,9,12 or 6) Eicosatrienoate 20:3 ( 8,11,14 or 6) desaturation Arachidonate 20:4 ( 5,8,11,14 or 6) elongation

11 Synthesis of Polyunsaturated Fatty Acids

12 Regulation of Fatty Acid Biosynthesis

13 Acetyl CoA Carboxylase Allosteric regulation citrate activates and palmitoyl CoA, stearoyl CoA and arachidyl CoA inhibit ACC Hormonal regulation glucagon inactivates and insulin activates ACC via phosphorylation/dephosphorylation of the enzyme

14 Regulation of ACC by Phosphorylation Inactivation (starved state) glucagon increases camp activates protein kinase A inactivates ACC Activation (fed state) insulin induces protein phosphatase and activates ACC

15 Regulation of Lipid Biosynthesis via SREBP Horton et al., J. Clin. Invest. 109: 1125, 2002.

16 SREBP1c Enhances transcription of genes required for FA synthesis Responsive genes include: ATP citrate lyase Acetyl-CoA carboxylase Fatty acid synthase Fatty acid elongase Stearoyl-CoA desaturase Glycerol-3-phosphate acyltransferase Genes required to generate NADPH

17 Insulin Stimulates hepatic FA synthesis during carbohydrate excess Increases SREBP-1c mrna in liver Induction of target genes blocked if dominant negative form of SREBP-1c is expressed Incubating primary hepatocytes with glucagon decreases mrnas for SREBP-1c and its associated target genes

18 What happens to these newly synthesized fatty acids? Depends on the cell and metabolic state Can be stored (but not as fatty acids) or utilized for energy Synthesis of other lipid classes

19 Glycerol backbone with 3 fatty acids (FA) Highly concentrated source of energy, easily stored in cytosolic droplets Before use as energy source, it must undergo lipolysis to release FA Triglyceride

20 Triglyceride Synthesis

21 Synthesis of Glycerol Phosphate in Liver Glucose Glycolysis DHAP CH 2 OH C=O O CH 2 -O-P-O - O NADH Glycerol 3-phosphate dehydrogenase NAD + CH 2 OH HO-C-H O CH 2 -O-P-O - O ADP ATP CH 2 OH CHOH CH 2 OH Glycerol Kinase Glycerol L-Glycerol 3-Phosphate

22 Biosynthesis of Triglyceride

23 What Happens to TG Synthesized by the Liver? Energy Export as VLDL Storage

24 Lipoprotein Model

25 Very Low Density Lipoproteins (VLDL) Spherical TG-rich particles nm in diameter Synthesized by the liver and transport triglyceride to periphery for storage and/or utilization Major structural protein is apo B

26 Apolipoprotein B Structural apoprotein for triglyceride-rich lipoproteins Expressed in two forms: apob100 & apob48 B100 is found on VLDL, IDL, and LDL B48 is found on chylomicrons ApoB100 and B48 synthesis are essential for VLDL and chylomicron assembly

27 Apolipoprotein B

28 VLDL Assembly Initiated in endoplasmic reticulum (ER) Assembly and secretion are dependent on the ability of the cell to synthesize apob and MTP Constitutive process modulated by a variety of factors

29 Assembly of Triglyceride-Rich Lipoproteins G. Shelness, Wake Forest School of Medicine

30 Microsomal Triglyceride Transfer Protein (MTP) A heterodimeric protein complex consisting of 97 kda subunit and protein disulfide isomerase (PDI) Transfers lipid from the ER membrane to the forming lipoprotein in the ER lumen Absolute requirement for assembly of VLDL by the liver Inhibition of MTP leads to reduction in plasma triglycerides and cholesterol

31 Regulation of VLDL Production Availability of functional MTP Apo B degradation Substrate availability

32 Functional MTP Inhibition of MTP leads to decreased VLDL secretion and a reduction in plasma TGs Over-expression of MTP leads to increased VLDL secretion Absence of MTP (abetalipoproteinemia) leads to absence of TG-rich lipoproteins in plasma

33 ApoB Degradation Proteasome inhibitors block degradation of apob and ubiquitinated apob accumulates within the cell ALLN, a calpain inhibitor, inhibits degradation of apob, but does not increase apob secretion Insulin regulates degradation of apob through PI3-kinase pathway Insulin resistance leads to decreased apob degradation and increased secretion

34 Assembly of Triglyceride-Rich Lipoproteins Davidson and Shelness, Ann. Rev. Nutr. 20: 169, 2000

35 Targets for Regulation of VLDL Assembly G.F. Gibbons, et al., Biochem Soc Trans 32: 59-64, 2004

36 Hepatic Lipid Mobilization Gibbons et al., Biochim. Biophys. Acta 1483: 37, 2000

37 What is the fate of VLDL secreted from the liver?

38 VLDL Metabolic Pathway

39 Chylomicron Metabolic Pathway

40 Adipocyte Lipid Metabolism Sethi J.K., Vidal-Puig A.J., J. Lipid Res. 48: , 2007

41 Sources of Fatty Acids for Liver and VLDL Triglycerides Adiels, M. et al. Arterioscler Thromb Vasc Biol 28: , 2008.

42 Why Does Liver Store TG? TG accumulates when plasma NEFA exceeds hepatic disposal via secretion and oxidation Neutralizes potential toxicity of fatty acids released from adipose tissue Hepatic TG secretion in post-absorptive state exceeds hepatic NEFA esterification, suggesting utilization of hepatic TG stores

43 Insulin A Key Regulator of Hepatic Lipid Metabolism Fatty acid flux to the liver Hepatic de novo lipogenesis MTP synthesis ApoB degradation Stimulates production of lipoprotein lipase Fatty acid oxidation

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45 De Novo Lipogenesis Insulin stimulates SREBP-1c expression and increases cleavage of SREBP-1c to its mature form which activates production of lipogenic enzymes Hepatic IR exists in the pathway regulating gluconeogenesis, but this does not affect insulin s ability to stimulate lipogenesis ChREBP is also up-regulated by insulin and works in synergy with SREBP-1c to promote lipogenesis

46 MTP Synthesis Mttp gene expression is negatively regulated by insulin in part by MAPK pathway Suppression of MTP by insulin also occurs via Forkhead box 01 (Fox01) Insulin induces phosphorylation of Fox01 leading to its exclusion from the nucleus, resulting in inhibition of MTP expression Fox01 gain of function is associated with enhanced MTP expression Insulin resistance leads to increased MTP expression

47 Insulin Signaling through Fox01 Kamagate et al. J. Clin. Invest. 118: , 2008

48 Degradation of ApoB Insulin targets apob for degradation via a PI3- kinase pathway Acute increases in insulin decrease hepatic VLDL and apob secretion via a post-er, non-proteasomal process Insulin resistance leads to decreased apob degradation and may contribute to overproduction of VLDL

49 Hepatic VLDL Assembly and Secretion Sparks and Sparks, J. Clin. Invest. 118: , 2008

50 Ginsberg et al., Arch. Med. Res. 36: 232, 2005 Substrate Sources Driving Assembly of ApoB-Lipoproteins in IR

51 Insulin, VLDL Secretion, and Fatty Liver Ginsberg, et al., Endocrin. Metab. Clinics 35: 491, 2006

52 Summary De novo lipogenesis and the regulation of fatty acid synthesis Sources of fatty acids for liver TG biosynthesis Secretion of hepatic TG with VLDL and the fate of TG-rich particles Insulin resistance and its impact on hepatic TG homeostasis

53 Effects of Insulin Resistance on Hepatic TG Homeostasis Increased fatty acid flux from adipose tissue to the liver Increased hepatic uptake of VLDL, IDL, and chylomicron remnants Increased de novo lipogenesis Decreased degradation of apob Increased activity of MTP Overproduction of VLDL Hepatic steatosis

54 Questions?

55 Increased Fatty Acid Flux from Adipose Tissue to the Liver Insulin stimulates adipocyte fatty acid uptake and inhibits fatty acid release (HSL) leading to decreased plasma NEFA Insulin resistance leads to increased mobilization of fatty acids from adipose tissue and increased plasma NEFA

56 De Novo Lipogenesis Hepatic insulin resistance leads to upregulation of sterol regulatory element-binding protein-1 (SREBP-1c) and activates lipogenic enzymes Carbohydrate response element-binding protein (ChREBP) regulates expression of key glucoseresponsive genes of lipogenesis Synergistic action of SREBP-1c and ChREBP directs conversion of excess glucose to fatty acids and enhances esterification

57 Substrate Sources Driving Assembly of ApoB-Lipoproteins in IR Ginsberg et al., Arch. Med. Res. 36: 232, 2005

58 Endocrine Reviews 20(5):

59 Changes in Lipoprotein Metabolism in the Metabolic Syndrome Adiels, M. et al. Arterioscler Thromb Vasc Biol 2008;28:

60 Metabolic Syndrome Abdominal obesity Elevated blood pressure Insulin resistance Prothrombotic state Proinflammatory state Atherogenic dyslipidemia

61 Metabolic Syndrome Dyslipidemia Elevated plasma triglycerides Decreased HDL cholesterol Normal levels of LDL cholesterol carried in small dense particles Lipoprotein alterations contribute to increased risk for CHD

62 Lecture Outline Lipoproteins, structure, apoproteins, characteristics Source of triglyceride for lipoproteins Assembly of triglyceride-rich lipoproteins Triglyceride-rich lipoprotein catabolism Effects of insulin resistance on triglyceride-rich lipoprotein production VLDL secretion and fatty liver

63 Hormonal control of adipocyte lipolysis Biochem. Soc. Trans. (2003) 31,

64 Apolipoproteins Lipoprotein Class Tissue Source MW apoai HDL 30K apoaii HDL liver and intestine 17K apoa-iv HDL 45K apob48 chylomicrons intestine 240K apob100 VLDL, LDL liver 512K apoci HDL 6.6K apocii HDL and VLDL liver 8.9K apociii HDL and VLDL 8.8K apoe HDL, LDL, VLDL liver 34K

65 Apolipoprotein B Structural apoprotein for triglyceride-rich lipoproteins Expressed in two forms: apob100 & apob48 B100 is found on VLDL, IDL, and LDL B48 is found on chylomicrons ApoB100 and B48 synthesis are essential for VLDL and chylomicron assembly

66 Increased MTP Activity Mttp gene has insulin response element in the promoter In human liver cells Mttp gene expression is negatively regulated by insulin through the MAPK cascade Increased MTP mrna is associated with enhanced synthesis of VLDL in wild-type animals and in animal models of insulin resistance

67 Increased MTP Activity Forkhead box 01 (Fox01) has been shown to mediate inhibitory action of insulin on target gene expression Fox01 stimulates hepatic MTP expression; the effect is counteracted by insulin Fox01 gain-of-function is associated with enhanced MTP expression, augmented hepatic VLDL production, and elevated plasma TG levels in Fox01 transgenic mice

68 Essential Fatty Acids (EFA) Fatty acids that cannot be synthesized and must be obtained from the diet C18:2-6 - linoleic acid C18: linolenic acid These fatty acids are starting point for synthesizing longer and more highly unsaturated fatty acids

69 Arachidonic Acid C20:4 ω6 COOH CH 3 A precursor for prostaglandins, leukotrienes, and thromboxanes

70 Common Fatty Acids C14:0 - myristic acid C16:0 - palmitic acid C18:0 - stearic acid C18:1 9 - oleic acid C18:2 6 - linoleic acid* C18: linolenic acid*; 6 - -linolenic acid C20:4 6 - arachidonic acid C20:5 3 - eicosapentaenoic acid C22:5 3 - docosapentaenoic acid C22:6 3 - docosahexaenoic acid

71 Source of Acetyl CoA Derived primarily from pyruvate via pyruvate dehydrogenase in mitochondria Transported into cytosol as citrate, then regenerated by ATP citrate lyase Available for malonyl CoA formation and fatty acid synthesis

72 CH 2 OH HO-C-H O CH 2 -O-P-O - O L-Glycerol 3-Phosphate O R 1 -C-S-CoA Acyl Transferase O R 2 -C-S-CoA Acyl Transferase CH 2 -OOCR 1 R 2 COO-C-H O CH 2 -O-P-O - O Phosphatidic Acid Addition of Fatty Acids to Form Triglycerides Phosphatidate phosphohydrolase CH 2 -OOCR 1 R 2 COO-C-H CH 2 OOCR 3 Triacylglycerol O R 3 -C-S-CoA DGAT CH 2 -OOCR 1 R 2 COO-C-H CH 2 OH Diacylglycerol

73 Plasma Lipoprotein Classes

74 Chylomicrons TG-rich particles 75 to 200 nm in diameter Assembled by enterocytes to transport dietary fat to periphery for storage or utilization Present in postprandial plasma ApoB-48 is the sole B apoprotein (humans and rodents)

75 Negative Stain of Lipoproteins Chylomicrons VLDL LDL HDL

76 Hepatic TG Storage Human liver can store 5-45 mol/g under normal conditions ( g/1.5 kg liver) Hepatic TG in house musk shrew can exceed 100 mol/g during starvation Livers of mice that overexpress SREBP- 1a contain >250 mol/g Liver accomodates TG in cytosolic droplets

77 Adipocyte Lipid Metabolism Sethi J.K., Vidal-Puig A.J., J. Lipid Res. 48: , 2007