Core Concepts
The following article will discuss ketogenesis, a metabolic pathway responsible for producing ketones, which provide an alternate source of energy in the body.
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Overview of Ketogenesis
Ketogenesis is a metabolic pathway responsible for the production of ketone bodies, which serve as an alternate energy substrate. This mechanism is crucial to the human body when in states of starvation as it allows stored fats to act as an energy source until more food energy becomes available. Ketogenesis occurs in the body at all times, but may be expedited in certain conditions. You may already be indirectly familiar with ketogenesis through the popular “keto” diet. The keto diet promotes ketogenesis by drastically reducing carbohydrate intake (or even completely omitting carbs from the diet). In the absence of carbohydrates (and therefore glucose) the body must rapidly burn fat to produce ketone bodies as an energy source. This expedited fat burn is what people aim to achieve in doing a keto diet. However, although ketogenesis is the body’s natural mechanism for providing an alternate fuel source, being in this state long-term is extremely dangerous. Ketone bodies are acidic, therefore, if too many accumulate in the body over long periods of time, blood pH is lowered and becomes toxic to the rest of the body. This phenomenon is called ketoacidosis and is fatal.
The Steps of Ketogenesis
Signaling
Ketogenesis occurs at all times in a healthy individual, however, certain conditions may cause ketone bodies to form at a faster rate. These conditions include low blood glucose levels, exhaustion of all available carbohydrates, or insufficient insulin levels (seen in Type 1 Diabetics). These conditions are also known as “the starving state”. On a molecular level, this is due to low insulin levels and high glucagon levels caused low blood glucose. This hormonal shift activates CPT-1 (carnitine palmitoyltransferase 1), an enzyme found primarily in the liver, whose main function is to catalyze the conversion and transport of fatty acids out of the liver into the mitochondria where they can be turned into an alternate energy source (ketone bodies). The activation of CPT-1 initiates the process of ketogenesis.
Transfer of Fatty Acids via CPT-1
Once ketogenesis is initiated, stored fatty acids in the liver must be transferred to the mitochondria. These fatty acids cannot cross the mitochondrial membrane, so the first step involves converting them to a form that is capable of crossing this membrane. This process begins with the conversion of fatty acids into fatty acyl-CoA molecules via acyl-CoA synthetase, an enzyme found in the cytosol. Then, CPT-1, located on the outer mitochondrial membrane, catalyzes the transfer of the fatty acyl group from CoA to carnitine, forming acylcarnitine. This transformation is crucial because acylcarnitine is now capable of crossing the otherwise impermeable inner mitochondrial membrane. The acylcarnitine is then shuttled across the inner mitochondrial membrane by a translocate enzyme known as carnitine-acylcarnitine translocate (CACT). Once inside the mitochondrial matrix, CPT-2 (carnitine palmitoyltransferase 2) reconverts acylcarnitine back into fatty acyl-CoA and a free carnitine. This reaction releases the free carnitine back into the mitochondrial inter membrane space, where it can be reused by CPT-1. The regenerated fatty acyl-CoA then enters the β-oxidation pathway.
β-Oxidation of Fatty Acid
In the β-oxidation step of ketogensis, a series of reactions occur cleaving two-carbon units from the fatty acid chain, converting them into acetyl-CoA. The process begins with the activation and transport of fatty acyl-CoA seen in the previous step. Once in the mitochondria, acyl-CoA dehydrogenase catalyzes the formation of a double bond between the second and third carbon atoms, forming trans-enoyl-CoA. This reaction also reduces FAD to FADH2, which later contributes to the electron transport chain. The trans-enoyl-CoA is then hydrated by enoyl-CoA hydratase, adding a molecule of water across the double bond to produce L-3-hydroxyacycl-CoA. Next, L-3-hydroxyacly-CoA dehydorgenase catalzes the oxidation of the hydroxyl group to a keto group, forming 3-ketoacyl-CoA. This step reduces NAD+ to NADH, another crucial electron carrier for the electron transport chain. In the final step, 3-ketoacyl-CoA is cleaved by β-ketothiolase, using a molecule of CoA to release one acetyl-CoA and a fatty acyl-CoA shortened by two carbon atoms.
In short, each cycle of β-oxidation generates one molecule of acetyl-CoA, along with reducing equivalents NADH and FADH₂. These reduced equivalents are essential for ATP production through oxidative phosphorylation in the mitochondria. The acetyl-CoA produced can either enter the citric acid cycle for typical energy production or, during starving states, serve as a substrate for the next step of ketogenesis.
Acetoacetyl-CoA Formation
In this step of ketogenesis, two molecules of acetyl-CoA combine to produce acetoacetyl-CoA through a condensation reaction. This reaction is catalyzed by a thiolase enzyme (acetoacetyl-CoA thiolase), which is localized within the mitochondria of hepatocytes (liver cells). This reaction is driven by the increased availability of acetyl-CoA resulting from the β-oxidation step seen previously. First, thiolase catalyzes the condensation of two acetyl-CoA molecules. The first acetyl-CoA molecule donates its carbonyl carbon, which acts as an electrophile, while the methyl group of the second acetyl-CoA acts as a nucleophile, attacking the carbonyl carbon of the first molecule. This nucleophilic attack forms a carbon-carbon bond between the two acetyl groups, yielding a four carbon intermediate: acetoacetyl-CoA and the release of coenzyme A. The acetoacetyl-CoA produced in this step serves as a critical precursor for the synthesis of HMG-CoA in the subsequent step.
HMG-CoA Synthesis
The next step of ketogenesis is 3-hydroxy-3-methylgutaryl-CoA (HMG-CoA) synthesis. This step occurs within the mitochondrial matrix and involves the condensation of acetyl-CoA and acetoacetyl-CoA molecules. The HMG-CoA synthase enzyme first binds to acetoacetyl-CoA, positioning it in its active site. The enzyme then binds to a molecule of acetyl-CoA. The acetyl-CoA donates an acetyl group, with its methyl group acting as a nucleophile. This nucleophile attacks the carbonyl carbon of the acetoacetyl-CoA, resulting in the formation of an enzyme bound intermediate. This intermediate then undergoes rearrangement, leading to the formation of a new carbon-carbon bond between the acetyl group and the β-keto group. This rearrangement results in the creation of β-hydroxy-β-methylglutaryl-CoA. During the final step, Coenzyme A is released from the intermediate complex. The thioester bond is cleaved, resulting in the final product, HMG-CoA. HMG-CoA serves as a precursor to acetoacetate, one of the primary ketone bodies formed during ketogenesis.
Acetoacetate Formation
The final step in ketogenesis is the formation of acetoacetate and occurs again in the mitochondrial matrix. This step is crucial in producing the primary ketone bodies used as an energy source in starving states. First, HMG-CoA binds to the active site of the enzyme HMG-CoA lyase. The enzyme positions the substrate so that the bond to be cleaved is optimally aligned. A hydrolysis reaction ensues where water acts as a nucleophile, attacking the carbonyl carbon, and cleaving the carbon-carbon bond between the β-hydroxy-β-methylglutaryl-CoA and the CoA moiety. This releases acetyl-CoA and acetoacetate.
Acetoacetate is one of the primary ketone bodies produced during ketogenesis. It can further undergo either non-enzymatic decarboxylation or enzymatic conversion to form other ketone bodies. In the case of decarboxylation, acetoacetate spontaneously decarboxylates and forms acetone and carbon dioxide. This reaction is relatively slow and occurs to a limited extent in the body. Enzymatically, acetoacetate can be reduced to D-β-hydroxybutyrate by the enzyme D-β-hydroxybutyrate dehydrogenase. This reaction involves reducing the ketone group in acetoacetate into a hydroxyl group, using NADH as a reducing agent. The rate of this reaction occurring depends on the availability of NADH in the cell.