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Lecture 20: Fatty Acid and Lipid Biosynthesis

Biochemistry

Learning Objectives

By the end of this lecture, students will be able to:

1. Describe the transport of acetyl-CoA from mitochondria to cytoplasm via the citrate shuttle
2. Explain the role and regulation of acetyl-CoA carboxylase (ACC)
3. Describe the reactions catalyzed by fatty acid synthase (FAS) and the role of ACP
4. Compare and contrast fatty acid synthesis with beta-oxidation
5. Describe the elongation and desaturation of fatty acids
6. Outline the synthesis of triacylglycerols, phospholipids, and cholesterol

Lecture Content

I. Overview of Fatty Acid Synthesis

Fatty acid synthesis takes place in the cytoplasm, primarily in the liver, adipose tissue, and lactating mammary gland. It occurs during the fed state, when energy and carbon are abundant. The starting material is acetyl-CoA, derived from glucose metabolism via pyruvate, and the end product is palmitate (C16:0), a 16-carbon saturated fatty acid. The pathway requires NADPH (supplied by the pentose phosphate pathway and the malic enzyme reaction), ATP, and CO2 in the form of bicarbonate.

It is important to recognize that fatty acid synthesis is NOT simply the reverse of beta-oxidation. The two pathways differ in virtually every dimension: cellular location (cytoplasm versus mitochondria), acyl carrier (ACP versus CoA), electron carrier (NADPH versus NADH and FADH2), the enzymes involved, and the two-carbon building block used (malonyl-CoA, a three-carbon intermediate that loses one carbon as CO2, versus acetyl-CoA released directly during degradation). These differences allow the cell to regulate synthesis and degradation independently.

II. Transport of Acetyl-CoA to the Cytoplasm: The Citrate Shuttle

Acetyl-CoA is produced in the mitochondrial matrix but cannot cross the inner mitochondrial membrane directly. The citrate shuttle (also called the tricarboxylate transport system) solves this problem through a series of steps. First, citrate synthase condenses acetyl-CoA with oxaloacetate to form citrate within the matrix. Citrate is then transported to the cytoplasm via the citrate transporter, which operates as an antiport with malate. In the cytoplasm, ATP-citrate lyase cleaves citrate in the presence of CoA and ATP, regenerating acetyl-CoA and oxaloacetate. Cytoplasmic malate dehydrogenase then reduces OAA to malate using NADH. The malic enzyme subsequently oxidatively decarboxylates malate to pyruvate and CO2, generating one molecule of NADPH that can be used for fatty acid synthesis. Finally, pyruvate returns to the mitochondria via the pyruvate carrier. The net effect of the citrate shuttle is therefore twofold: it transfers acetyl-CoA to the cytoplasm and generates NADPH in the process.

III. Acetyl-CoA Carboxylase (ACC) — The Committed Step

Acetyl-CoA carboxylase catalyzes the rate-limiting and committed step of fatty acid synthesis, converting acetyl-CoA, bicarbonate, and ATP into malonyl-CoA, ADP, and inorganic phosphate. The enzyme requires biotin as a covalently attached prosthetic group that serves as a CO2 carrier. The reaction proceeds in two steps: first, biotin is carboxylated using ATP and bicarbonate; then the carboxyl group is transferred from biotin to acetyl-CoA, forming malonyl-CoA.

Regulation of ACC:

ACC is regulated at multiple levels. Allosteric regulation plays a key role: citrate activates the enzyme, signaling that building blocks are abundant, while palmitoyl-CoA inhibits it through end-product feedback. Covalent modification provides additional control. AMPK (AMP-activated protein kinase) phosphorylates ACC to render it inactive when cellular energy is low, while protein phosphatase 2A dephosphorylates and activates ACC when energy is plentiful. At the hormonal level, glucagon and epinephrine activate AMPK and thereby inactivate ACC, decreasing fatty acid synthesis, while insulin activates the phosphatase and promotes ACC activity and fatty acid synthesis. Transcriptional regulation adds a longer-term layer of control: insulin and a high-carbohydrate diet upregulate ACC gene expression through the transcription factor SREBP-1c, whereas fasting and glucagon downregulate expression.

IV. Fatty Acid Synthase (FAS)

In mammals, fatty acid synthase is a single large multifunctional polypeptide (type I FAS) that functions as a homodimer, with each monomer weighing approximately 270 kDa and containing seven distinct enzymatic activities plus an acyl carrier protein (ACP) domain. The ACP bears a phosphopantetheine arm (derived from CoA) that carries the growing acyl chain throughout the synthesis cycle.

The Reactions of FAS

The FAS reaction cycle consists of a loading phase followed by four repeating reactions. During loading, an acetyl group from acetyl-CoA is placed onto the Cys-SH of the ketoacyl synthase (KS) domain, and a malonyl group from malonyl-CoA is loaded onto the ACP phosphopantetheine arm by the malonyl/acetyl transferase.

Step 1, Condensation (Ketoacyl Synthase), joins the acetyl group and the malonyl group to form acetoacetyl-ACP, releasing CO2. This CO2 is the same carbon that was added by ACC, and its release provides the thermodynamic driving force for condensation. Each cycle extends the chain by two carbons. Step 2, Reduction (Ketoacyl Reductase), reduces the keto group to a hydroxyl using NADPH, producing D-3-hydroxybutyryl-ACP. Step 3, Dehydration (Hydroxyacyl Dehydratase), removes water to introduce a trans double bond, yielding crotonyl-ACP. Step 4, Reduction (Enoyl Reductase), reduces the double bond to a single bond using a second NADPH, producing butyryl-ACP.

After one complete cycle, the four-carbon butyryl group is transferred back to the KS Cys-SH, and a new malonyl-CoA is loaded onto ACP. The cycle repeats six more times for a total of seven cycles. After seven cycles, the product is a 16-carbon palmitoyl-ACP. The thioesterase (TE) domain then cleaves palmitate from ACP, releasing free palmitate.

The overall equation is: acetyl-CoA + 7 malonyl-CoA + 14 NADPH + 14 H+ yields palmitate + 7 CO2 + 14 NADP+ + 8 CoA + 6 H2O. The total cost is 8 acetyl-CoA + 7 ATP + 14 NADPH.

<image>A diagram of the fatty acid synthase reaction cycle. Panel A: The structure of the FAS homodimer as a schematic showing the arrangement of the seven enzymatic domains (MAT, KS, KR, DH, ER, TE) and the ACP arm with phosphopantetheine. Panel B: The four-step reaction cycle shown as a circular pathway: condensation (KS), first reduction (KR, NADPH), dehydration (DH), and second reduction (ER, NADPH). The growing chain is extended by 2 carbons per cycle. Panel C: An overview showing acetyl-CoA as the primer, 7 malonyl-CoA additions, and the final release of palmitate (C16) by thioesterase. The sources of NADPH (pentose phosphate pathway and malic enzyme) are indicated.</image>

V. Comparison: Fatty Acid Synthesis vs. Beta-Oxidation

VI. Elongation and Desaturation

Fatty Acid Elongation

Further elongation of palmitate beyond 16 carbons occurs primarily in the smooth ER (and to a lesser extent in mitochondria). The chemistry resembles that of FAS, using malonyl-CoA and NADPH, but distinct enzymes carry out the reactions. This pathway extends palmitate (C16) to stearate (C18), arachidonate (C20), and longer chains.

Fatty Acid Desaturation

The introduction of cis double bonds into fatty acyl chains is accomplished by fatty acid desaturases, which are mixed-function oxidases located in the smooth ER. These enzymes require molecular oxygen, NADH, and cytochrome b5 reductase. Mammals possess delta-9, delta-6, delta-5, and delta-4 desaturases. Critically, mammals cannot introduce double bonds beyond carbon 9 when counting from the carboxyl end. This means that omega-3 and omega-6 fatty acids cannot be synthesized de novo, making linoleic acid (18:2, omega-6) and alpha-linolenic acid (18:3, omega-3) essential fatty acids that must be obtained from the diet. Arachidonic acid (20:4, omega-6) can be synthesized from linoleic acid by a combination of elongation and desaturation and serves as the precursor for eicosanoids, including prostaglandins, thromboxanes, and leukotrienes.

VII. Triacylglycerol Synthesis

Triacylglycerol synthesis occurs in the liver and adipose tissue. The pathway begins with glycerol-3-phosphate, which can be generated from DHAP (via glycerol-3-phosphate dehydrogenase) or from glycerol (via glycerol kinase, present only in the liver). Two sequential acylations with acyl-CoA produce phosphatidic acid (PA). Dephosphorylation of PA by phosphatidic acid phosphatase (lipin) yields diacylglycerol (DAG), and a third acylation with acyl-CoA converts DAG to triacylglycerol (TAG). In the liver, TAG is packaged into VLDL particles for export to peripheral tissues, whereas in adipose tissue, TAG is stored in lipid droplets.

VIII. Phospholipid Synthesis

Phosphatidic acid serves as the central intermediate for phospholipid synthesis. Two main pathways diverge from this point. The CDP-diacylglycerol pathway converts PA to CDP-DAG, which then gives rise to phosphatidylinositol (PI), phosphatidylglycerol (PG), and cardiolipin. The Kennedy pathway proceeds via DAG: DAG combined with CDP-choline yields phosphatidylcholine (PC), and DAG combined with CDP-ethanolamine yields phosphatidylethanolamine (PE). PE can be methylated to PC by the enzyme PEMT, using SAM as the methyl donor, primarily in the liver. Phosphatidylserine (PS) is produced by head-group exchange from PE or PC.

IX. Cholesterol Synthesis — Overview

Cholesterol synthesis takes place in the cytoplasm and ER, primarily in the liver. All 27 carbon atoms of cholesterol derive from acetyl-CoA. The rate-limiting enzyme is HMG-CoA reductase, which converts HMG-CoA to mevalonate using two molecules of NADPH. This enzyme is the target of statins, which act as competitive inhibitors and effectively lower serum cholesterol levels. Mevalonate is subsequently converted through several steps to isopentenyl pyrophosphate (an activated isoprene unit), then to squalene, lanosterol, and finally cholesterol.

Cholesterol synthesis is tightly regulated. When intracellular cholesterol levels are low, SREBP (sterol regulatory element-binding protein) is activated as a transcription factor, increasing expression of both HMG-CoA reductase and the LDL receptor. Cholesterol and oxysterols exert feedback inhibition at multiple levels when they accumulate. Insulin activates cholesterol synthesis while glucagon inhibits it, consistent with the general pattern of anabolic versus catabolic hormonal signaling.

<image>A summary diagram of lipid biosynthesis showing the interconnected pathways. Central panel: Acetyl-CoA as the starting point, with arrows to the citrate shuttle, then ACC producing malonyl-CoA, then FAS producing palmitate. From palmitate, arrows show elongation (to longer chains) and desaturation (to unsaturated fatty acids). Branching pathways show: glycerol-3-phosphate + acyl-CoA leading to phosphatidic acid, which branches to triacylglycerols (for storage) and phospholipids (for membranes). A separate branch shows acetyl-CoA to HMG-CoA to mevalonate to cholesterol, with statins inhibiting HMG-CoA reductase. Key regulatory points are marked: ACC (committed step for FA synthesis, regulated by citrate, palmitoyl-CoA, AMPK) and HMG-CoA reductase (committed step for cholesterol, regulated by statins and SREBP).</image>

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