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Role of Liver In Triglyceride Homeostasis
Larry L. Swift, Ph.D.
Department of Pathology
Vanderbilt University School of Medicine
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)
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 18:3 ω6
Eicosapentaenoic 20:5 ω3
Arachidonic 20:4 ω6
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
Fatty acidsAmino Acids
Glucose
Pyruvate
Denhydrgenase
Acetyl CoA
Fatty Acids
Cytosol
Pyruvate
Mitochondria
Fatty Acid Biosynthesis
•First committed step of fatty acid synthesis pathway
•Acetyl CoA Carboxylase (ACC) is major regulatory site of fatty acid synthesis
Fatty Acid Elongation / Desaturation
Palmitate (16:0)
Palmitoleate
16:1 (∆9 or 7)Stearate (18:0)
Longer saturated
fatty acidsOleate
18:1 (∆9 or 9)
Linoleate
18:2 (∆9,12 or 6)-linoleate
18:3 (∆6,9,12 or 6)
desaturation
desaturation
elongation
Eicosatrienoate
20:3 (∆8,11,14 or 6)
Arachidonate
20:4 (∆5,8,11,14 or 6)
elongation
desaturation
-linoleate
18:3 (∆9,12,15 or 3)
Other PUFAs
Mammals
and plants
* Plants
only
*
*
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
• Inactivation (starved state)– glucagon increases
cAMP– activates protein
kinase A– inactivates ACC
• Activation (fed state)– insulin induces
protein phospha-tase and activates ACC
Regulation of ACC by Phosphorylation
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
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
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
Triglyceride
• 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
Synthesis of Glycerol Phosphate in Liver
L-Glycerol 3-Phosphate
Glycolysis
Glucose
Glycerol 3-phosphate
dehydrogenase
NADH
NAD+
CH2OH
C=O
O
CH2-O-P-O-
O
Glycerol
ATP
ADP
Glycerol Kinase
CH2OH
CHOH
CH2OH
CH2OH
HO-C-H
O
CH2-O-P-O-
O
DHAP
Very Low Density Lipoproteins (VLDL)
• Spherical TG-rich particles 40-60 nm in diameter
• Synthesized by the liver and transport triglyceride to
periphery for storage and/or utilization
• Major structural protein is apo B
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
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
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
Regulation of VLDL Production
• Availability of functional MTP
• Apo B degradation
• Substrate availability
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
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
Adiels, M. et al. Arterioscler Thromb Vasc Biol 28:1225-1236, 2008.
Sources of Fatty Acids for Liver and VLDL Triglycerides
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
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
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
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
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
Substrate Sources Driving Assembly of ApoB-Lipoproteins in IR
Ginsberg et al., Arch. Med. Res. 36: 232, 2005
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
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
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
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 glucose-responsive genes of lipogenesis
• Synergistic action of SREBP-1c and ChREBP directs conversion of excess glucose to fatty acids and enhances esterification
Substrate Sources Driving Assembly of ApoB-Lipoproteins in IR
Ginsberg et al., Arch. Med. Res. 36: 232, 2005
Adiels, M. et al. Arterioscler Thromb Vasc Biol 2008;28:1225-1236
Changes in Lipoprotein Metabolism in the Metabolic Syndrome
Metabolic Syndrome
• Abdominal obesity
• Elevated blood pressure
• Insulin resistance
• Prothrombotic state
• Proinflammatory state
• Atherogenic dyslipidemia
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
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
Apolipoproteins
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
Lipoprotein
ClassTissue Source MW
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
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
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
Essential Fatty Acids (EFA)
• Fatty acids that cannot be synthesized and
must be obtained from the diet
• C18:2 -6 - linoleic acid
• C18:3 -3 - -linolenic acid
• These fatty acids are starting point for
synthesizing longer and more highly
unsaturated fatty acids
Arachidonic Acid
• C20:4 ω6
• A precursor for prostaglandins, leukotrienes,
and thromboxanes
COOH
CH3
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*
• C18:3 3 - -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
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
L-Glycerol 3-Phosphate
CH2OH
HO-C-H
O
CH2-O-P-O-
O
Acyl Transferase
O O
R1-C-S-CoA R2-C-S-CoA
CH2-OOCR1
R2COO-C-H
O
CH2-O-P-O-
O
Acyl Transferase
Phosphatidic Acid
CH2-OOCR1
R2COO-C-H
CH2OH
Diacylglycerol
O
R3-C-S-CoA
DGAT
CH2-OOCR1
R2COO-C-H
CH2OOCR3
Triacylglycerol
Addition of Fatty Acids to
Form Triglycerides
Phosphatidate
phosphohydrolase
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)
Hepatic TG Storage
• Human liver can store 5-45 mol/g under
normal conditions (6.7-60.3 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
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