Lipogenesis Fats not only obtained from the diet but also obtained from lipogenesis in the body. Lipogenesis means synthesis of neutral fats (TAG) from CHO and proteins present in excess of body need. Lipogenesis requires: 1- Synthesis of fatty acids (FA) and glycerol 2- Activation of fatty acids (by CoA) and glycerol (by glycerokinase) 3- the combination of activated fatty acids and activated glycerol to produce TAG.

1- De novo synthesis of fatty acid (cytoplasmic synthesis): Occur mainly for the synthesis of palmitic acid (C16:0) Site: Cytoplasm of liver, mammary glands and adipose tissues. Acetyl CoA is the precursor of fatty acid synthesis. It is produced from oxidation of glucose (by oxidative decarboxylation of pyruvate), and metabolism of ketogenic and mixed amino acids. In case of high CHO diet, excess acetyl CoA is produced. Some enter kreb’s and the excess will participate in fatty acid synthesis in cytoplasm of liver, mammary glands and adipose tissue.

Steps of fatty acid synthesis: 1- Transport of acetyl CoA to cytoplasm: Acetyl CoA is produced in mitochondria, and FA synthesis occurs in cytoplasm, so acetyl CoA must be transferred to cytoplasm across mitochondrial membrane which is impermeable to CoA. The transport occur through condensation of acetyl CoA inside mitochondria with oxaloacetate (OAA) to form citrate which can be transferred into cytoplasm. In cytoplasm, citrate is cleaved by ATP-citrate lyase or called citrate cleavage system in the presence of ATP and CoA to give acetyl CoA and OAA. Conclusion: Acetyl-CoA is delivered to cytosol from the mitochondria as CITRATE

-CoA ↓ citrate synthase Transport of acetyl CoA from mitochondria to cytoplasm: Mitochondria: OAA + Acetyl CoA -CoA ↓ citrate synthase Citrate Inner mitochondrial membrane ↓ + CoA, ATP ↓ ATP citrate lyase Cystosol

Transport of acetyl CoA from mitochondria to cytoplasm:

The rate limiting step in FA synthesis is the 2-Conversion of acetyl CoA into malonyl CoA by acetyl CoA carboxylase (ACC) The rate limiting step in FA synthesis is the synthesis of malonyl CoA by combination of acetyl CoA and CO2 in the presence of ATP This step is catalyzed by acetyl CoA carboxylase (ACC) which is the rate limiting enzyme. ACC contains biotin which is the carrier of CO2 [CO2 is covalently linked to biotin which is bound to lysine residue of ACC].

Reaction catalyzed by Acetyl CoA Carboxylase

3- Remaining series of the pathway is catalyzed by fatty acid synthase (FAS) which is a multifunctional enzyme. The pathway catalyzed by FAS involve 7 cycles to produce finally palmitate Cycle 1: The condensation of acetyl-CoA and two carbons derived from malonyl-CoA, with elimination of CO2 from the malonyl group forming at the end 4 carbon acyl (buteryl), Cycle 2: buteryl + malonyl CoA (with the elimination of CO2 from malonate) gives 6 carbon acyl, Cycle 3: 6C acyl + malonyl CoA gives 8 C acyl, and so on for a total 7 cycles ending with the synthesis of 16 C acyl (palmitate). In each cycle 2 carbon atoms are added. The source of these two carbons is malonyl CoA.

Fatty acid synthase (FAS) contains a phosphopantetheine residue called acyl carrier protein (ACP), derived from the vitamin pantothenic acid, and a cysteine residue; Both ACP and cystein rediude contain sulfhydryl (thiol) that can form thioesters with acyl groups.

Steps of FA synthesis by Fatty Acid Synthase (FAS): Initially, acetyl CoA reacts with the thiol of ACP and then the acetyl group is transferred to the thiol of cysteine residue. This acetyl group provides the ω-carbon of the fatty acid produced by the fatty acid synthase complex. A malonyl group from malonyl-CoA forms a thioester with the thiol of ACP Condensation: The acetyl group on the fatty acid synthase complex condenses with the malonyl group; the CO2 is released; and a β-ketoacyl group, containing four carbons, is produced. Reduction: The β-keto group is reduced by NADPH to a β-hydroxy group. Dehydration: Then dehydration occurs, producing an enoyl group with the double bond between carbons 2 and 3 Reduction: Finally, the double bond is reduced by NADPH, and a four-carbon acyl group is generated.

Overall reactions of FA synthesis: Acetyl-CoA + 7 Malonyl-CoA + 14 NADPH + H+ Palmitic acid (16:0) +7 CO2 + 8 CoA +14 NADP+ The ω carbon and its adjacent carbon are produced from acetyl CoA (from the first cycle), the reaming 14 carbons are derived from malonyl CoA (7 molecules; each molecule add 2 carbons in each cycle of the 7 cycles)

Role of NADPH in FA synthesis: NADPH is important hydrogen carrier for FA synthesis (HOW?) The condensation of acetyl-CoA and malonyl-CoA form β-keto acyl (not the acyl itself), so the keto group should be reduced to give the final product of the cycle which is the saturated acyl. The reduction steps needs NADPH+H as source of hydrogen that convert keto group to CH2

Sources of NADPH for fatty acid synthesis From where does NADPH needed for synthesis come? pentose phosphate pathway (HMP pathway) is the major source. Reduction of OAA (that is produced from citrate cleave system in the cytoplasm) to malate followed by conversion of malate to pyruvate by malic enzyme is a source of NADPH

Regulation of acetyl CoA carboxylase (ACC) or regulation of fatty acid synthesis: A) Allosteric regulation: allosterically activated by citrate & ATP and inhibited by the product of pathway (palmitic acid). B) Hormonal regulation: activated by insulin, inhibited by adrenaline and glucagon (anti-insulin hormone). Insulin lead to the formation of the active form of the ACC (dephosphorylated form) because insulin antagonize the action of cAMP/protein kinase activity. cAMP leads to activation of protein kinase A enzyme leading to phosphorylation of ACC which will be inactive form. Insulin activates a phosphatase enzyme called protein phosphatase 2A to dephosphorylate the enzyme; thereby activating it and remove the inhibitory effect of cAMP/protein kinase. C) Dietary regulation: prolonged consumption of high claoric diet (e.g CHO diet) increases the synthesis of acetyl CoA carboxylase and so increase FA synthesis. Fat-free diet, fasting and low CHO reduce enzyme synthesis and so decrease FA synthesis.

Regulation of ACC:

Elongation of fatty acids: Site: Mitochondria and endoplasmic reticulum Palmitic acid - the end product of FA synthesis in cytoplasm can be elongated in endoplasmic reticulum and mitochondria by the addition of two carbon atoms from malonyl CoA to give stearic acid and longer chain saturated FAs Palmitate is the precursor of stearic and longer-chain saturated fatty acids, as well as the monounsaturated acids (palmitoleic and oleic). Mammals cannot convert oleic to linoleic or linolenic (shaded pink) because humans cannot introduce additional double bonds from the C10 to the methyl-terminal (ω carbon). So, linoleic or linolenic are therefore required in the diet as essential fatty acids. Arachidonic acid is synthesized in the body from linolenic acid

Desaturation The fatty acid desaturation occurs in the endoplasmic reticulum - Humans (mammals) have four desaturase enzymes that add double bonds at carbons 4, 5, 6, 9 in mammals. And as we told in previous slide humans can’t add double bond to carbon 10 to terminal carbon, but plants can.