Chapter 9: Carbohydrate Metabolism I: Glycolysis and Gluconeogenesis

Metabolism is every chemical reaction happening in a cell. It splits into two directions: catabolism breaks complex molecules into simpler ones and harvests energy, and anabolism builds complex molecules from simpler ones using energy.

For this chapter and the next two, keep this simple map in your head for catabolism:

1. Glucose → glycolysis → pyruvate + 2 ATP + 2 NADH.
2. Pyruvate → PDH → acetyl-CoA + NADH + CO2.
3. Acetyl-CoA → TCA cycle (citric acid cycle / Krebs cycle) → 3 NADH + FADH2 + GTP + 2 CO2 per turn.
4. NADH and FADH2 → electron transport chain → proton gradient.
5. Proton gradient → ATP synthase → ATP.

Fatty acids and amino acids plug into the same pipeline at acetyl-CoA (or a TCA intermediate). The TCA cycle’s main job is not ATP - it is making NADH and FADH2 to feed the ETC. Think of the TCA cycle as the electron loader and the ETC as the ATP maker.

Catabolism vs. Anabolism

| Process | Direction | Energy | Examples |
|---------|-----------|--------|----------|
| Catabolism | Large → small | Releases energy (captured as ATP, NADH, FADH2) | Glycolysis, beta-oxidation, amino acid catabolism |
| Anabolism | Small → large | Consumes energy (ATP, NADPH) | Gluconeogenesis, fatty acid synthesis, protein synthesis |

Acetyl-CoA - The Central Hub

All major fuels converge to acetyl-CoA, which enters the TCA cycle:

• Glucose → glycolysis → pyruvate → pyruvate dehydrogenase → acetyl-CoA.
• Fatty acids → beta-oxidation → acetyl-CoA.
• Some amino acids → directly or via pyruvate → acetyl-CoA.

Energy Carriers

Cells store catabolic energy in three main molecules:

• ATP: immediate energy currency. Hydrolysis of ATP to ADP releases ~7.3 kcal/mol.
• NADH and FADH2: electron carriers. Deliver electrons to the ETC, which makes ATP.
• NADPH: like NADH but used for biosynthetic reductions (fatty acid synthesis, antioxidant regeneration) and made by the pentose phosphate pathway.

The first half of glycolysis consumes 2 ATP to prepare glucose for splitting and cleaves the resulting 6-carbon molecule into two 3-carbon molecules.

The Five Investment Steps

Key Points

• Step 1 (hexokinase): glucose enters the cell via GLUT transporters, then is phosphorylated. Once phosphorylated, glucose cannot leave the cell - it is committed. Hexokinase has a low Km and is in all cells. Liver uses glucokinase (high Km), which only activates at high blood glucose (post-meal).
• Step 3 (PFK-1): the committed, rate-limiting step. Heavily regulated by ATP (inhibits), AMP (activates), citrate (inhibits), and fructose-2,6-bisphosphate (strongly activates). Hormonal signals (insulin vs. glucagon) control F-2,6-BP levels via PFK-2.
• Step 4 (aldolase): splits the 6-carbon sugar into two 3-carbon sugars: DHAP and G3P.
• Step 5 (triose phosphate isomerase): DHAP is converted to G3P. So at the end of step 5, the cell has two G3P molecules, both of which will proceed through the payoff phase.

The payoff phase oxidizes each 3-carbon glyceraldehyde-3-phosphate to pyruvate, harvesting energy as NADH and ATP. Because every glucose produces 2 G3P, each payoff step runs twice per glucose.

The Five Payoff Steps

Each G3P runs through these five steps. Because one glucose produces two G3P, multiply every yield by 2 for the whole-glucose total.

Net Yield

Per molecule of glucose:

• 4 ATP produced (2 per G3P × 2 G3P) in the payoff
• 2 ATP consumed in the investment phase
• Net: 2 ATP
• Plus: 2 NADH and 2 pyruvate

Substrate-Level Phosphorylation

The two ATPs in the payoff phase are made by substrate-level phosphorylation - a high-energy phosphate group is transferred directly from a phosphorylated intermediate to ADP. This is different from oxidative phosphorylation (ETC + ATP synthase), which uses a proton gradient.

Arsenic Poisoning

Arsenite (As3+) inhibits the sulfhydryl groups needed by GAPDH. Arsenate (As5+) mimics phosphate, becomes incorporated at step 6 but then spontaneously hydrolyzes instead of phosphorylating ADP in step 7 - effectively uncoupling glycolysis without producing ATP. Both are lethal because glycolysis is required in all cells.

Three enzymes catalyze the irreversible steps of glycolysis and are the main regulatory points: hexokinase (step 1), PFK-1 (step 3), and pyruvate kinase (step 10). Of these, PFK-1 is the most heavily regulated and is called the master switch of glycolysis.

PFK-1: The Master Switch

PFK-1 is allosterically controlled by multiple effectors:

Fructose-2,6-Bisphosphate - The Hormonal Link

F-2,6-BP is not a glycolysis intermediate but a dedicated regulator. It is made by PFK-2 (a different enzyme from PFK-1) from F6P. PFK-2 has two activities: a kinase (makes F-2,6-BP) and a phosphatase (removes it). Insulin and glucagon control which activity dominates:

• Insulin (fed state): dephosphorylates PFK-2 → kinase activity dominates → F-2,6-BP goes UP → PFK-1 ACTIVATED → glycolysis speeds up.
• Glucagon (fasted state): phosphorylates PFK-2 → phosphatase activity dominates → F-2,6-BP goes DOWN → PFK-1 inhibited → glycolysis slows. Simultaneously, gluconeogenesis is activated.

This reciprocal regulation at the same control point (PFK-1) lets a single hormone shift metabolism between storage (fed) and mobilization (fasted).

Hexokinase and Pyruvate Kinase

• Hexokinase: inhibited by its product G6P (end-product inhibition). Glucokinase (liver version) has higher Km and is NOT inhibited by G6P, so the liver continues to phosphorylate glucose at high blood-glucose levels.
• Pyruvate kinase: activated by feed-forward signal F-1,6-BP (upstream intermediate); inhibited by ATP and alanine. In liver, pyruvate kinase is also phosphorylated (inactivated) by glucagon-triggered PKA, reducing pyruvate production during fasting.

Each pyruvate produced by glycolysis has three possible fates, depending on the presence of oxygen and the cell type. This is the branch point where glucose metabolism splits into “aerobic with full oxidation” or “anaerobic with fermentation.”

Fate 1: Acetyl-CoA (Aerobic) - The Main Pipeline

If oxygen is available, pyruvate enters the mitochondrial matrix and is oxidatively decarboxylated by the pyruvate dehydrogenase (PDH) complex:

$\text{Pyruvate + CoA + NAD}^+ \rightarrow \text{Acetyl-CoA + CO}_2 + \text{NADH}$

Each glucose produces 2 pyruvate → 2 acetyl-CoA + 2 CO2 + 2 NADH (in the mitochondrial matrix). Acetyl-CoA then enters the TCA cycle (also called the citric acid cycle or Krebs cycle) for full oxidation. The TCA cycle strips more electrons as NADH and FADH2, which feed the electron transport chain (ETC). The ETC pumps protons, and ATP synthase uses the proton gradient to make ATP. This is the main energy pipeline of aerobic respiration, covered in Chapter 10.

PDH requires 5 coenzymes: TPP (B1), FAD (B2), NAD+ (B3), CoA (B5), and lipoic acid. Mnemonic: “Tender Loving Care For Nancy” = TPP, Lipoic acid, CoA, FAD, NAD.

Fate 2: Lactate (Anaerobic in Animals)

When O2 is scarce or mitochondria are absent (red blood cells have no mitochondria), pyruvate is reduced to lactate:

$\text{Pyruvate + NADH} \xrightarrow{\text{lactate dehydrogenase}} \text{Lactate + NAD}^+$

Why bother? Glycolysis requires NAD+ to keep going (step 6, GAPDH). Without the ETC to recycle NADH back to NAD+, the NAD+ pool would run out. Making lactate regenerates NAD+ so glycolysis can continue (~2 ATP per glucose, slow but oxygen-independent).

Fate 3: Ethanol (Yeast Fermentation)

In yeast, pyruvate is decarboxylated to acetaldehyde, then reduced to ethanol:

$\text{Pyruvate} \rightarrow \text{Acetaldehyde + CO}_2 \rightarrow \text{Ethanol}$

The second step uses NADH, regenerating NAD+ just like lactic acid fermentation. This is how beer, wine, and bread dough are made - yeast ferment sugar, producing CO2 (bubbles) and ethanol (alcohol).

Gluconeogenesis is the synthesis of glucose from non-carbohydrate precursors. It happens mainly in the liver and (to a lesser extent) the kidney. It is essentially glycolysis in reverse, but with four different “bypass” enzymes at the three irreversible steps of glycolysis.

Gluconeogenesis connects to the other metabolic pathways by running them backwards. The glycerol backbone from fat (from triglyceride breakdown) and amino acids (from muscle protein) both feed into gluconeogenesis at different points. Lactate from anaerobic glycolysis in muscle and RBCs is shipped to the liver (Cori cycle) and converted back to glucose here. Essentially, gluconeogenesis is the liver’s reverse-engineering machine that makes glucose when we cannot get it from food.

Why Gluconeogenesis Matters

During fasting, brain and red blood cells still need glucose. Once glycogen stores are depleted (~24 hours), the body must make glucose from precursors. Gluconeogenesis ensures a steady blood-glucose supply even during prolonged fasting.

Precursors

• Lactate (from muscle / RBC glycolysis) → pyruvate → glucose. Cori cycle.
• Glycerol (from triglyceride breakdown) → DHAP → glucose.
• Glucogenic amino acids (especially alanine from muscle proteolysis) → pyruvate or TCA intermediates → glucose.

The Four Bypass Enzymes

Glycolysis has three irreversible steps (1, 3, 10). Gluconeogenesis cannot simply reverse them; it uses different enzymes to bypass.

Pyruvate to PEP

This two-step bypass of pyruvate kinase is the most energetically expensive part:

1. Pyruvate carboxylase (in mitochondrion) adds CO2 to pyruvate, making oxaloacetate. Uses 1 ATP. Requires biotin (vitamin B7).
2. PEP carboxykinase (PEPCK) decarboxylates oxaloacetate and phosphorylates it to PEP. Uses 1 GTP.

Pyruvate carboxylase is activated by acetyl-CoA - a signal that fat is being burned, so glucose is scarce, and the liver should synthesize more.

Glucose-6-Phosphatase

Only the liver and kidney have glucose-6-phosphatase. This enzyme removes the phosphate from G6P, producing free glucose that can leave the cell via GLUT2. Muscle lacks this enzyme - that is why muscle glycogen cannot directly contribute to blood glucose. Only liver glycogen can be broken down and released as blood glucose.

Cost

Per glucose synthesized from 2 pyruvate: 4 ATP + 2 GTP + 2 NADH consumed. Gluconeogenesis is expensive - the body only runs it when glucose is truly needed (fasting, low blood sugar, post-exercise).

Glycogen is the animal storage form of glucose - highly branched alpha-1,4 + alpha-1,6 polymer found in liver (for blood glucose buffering) and muscle (for local energy). Building it up and breaking it down are separate pathways, reciprocally regulated.

Glycogen metabolism is a short detour off of glycolysis. When the cell has excess glucose-6-phosphate (the first glycolysis intermediate), it can store it as glycogen rather than running it through glycolysis. When energy is needed later, glycogen is broken down back to glucose-6-phosphate, which can then re-enter glycolysis (in muscle) or be released as blood glucose after G6Pase action (in liver). So glycogen is a short-term reserve that feeds back into the glycolysis → TCA → ETC pipeline when the cell needs it.

Glycogenesis (Building)

1. Glucose-6-P → glucose-1-P (phosphoglucomutase).
2. G1P + UTP → UDP-glucose + PPi (UDP-glucose pyrophosphorylase). UDP-glucose is the activated donor.
3. Glycogen synthase adds UDP-glucose to the non-reducing end of a growing glycogen chain via alpha-1,4 linkage. Rate-limiting enzyme of glycogenesis.
4. When the chain is ~11 residues long, branching enzyme transfers a short segment to an internal position via an alpha-1,6 bond, creating a branch.

Glycogenolysis (Breaking Down)

1. Glycogen phosphorylase cleaves glucose-1-P from the non-reducing end of a branch using inorganic phosphate (Pi). Rate-limiting enzyme. Uses pyridoxal phosphate (PLP, vitamin B6) as a cofactor.
2. When phosphorylase reaches 4 residues from a branch point, it stops.
3. Debranching enzyme (a dual-function enzyme) moves three residues to the main chain (transferase activity) and then hydrolyzes the remaining alpha-1,6 glucose (glucosidase activity), releasing a free glucose.
4. G1P → G6P (phosphoglucomutase). In liver, glucose-6-phosphatase converts G6P → free glucose, which leaves the cell. In muscle, G6P enters glycolysis for local ATP.

Hormonal Regulation

Insulin (fed state):

• Dephosphorylates glycogen synthase → ACTIVE → storage.
• Dephosphorylates glycogen phosphorylase → INACTIVE → no breakdown.

Glucagon (fasted) and epinephrine (stress):

• Phosphorylates glycogen synthase → INACTIVE → no storage.
• Phosphorylates glycogen phosphorylase kinase → phosphorylates glycogen phosphorylase → ACTIVE → breakdown.

Both hormones work through cAMP/PKA cascades that phosphorylate multiple targets simultaneously.

Glycogen Storage Diseases

Mutations in glycogen enzymes cause characteristic storage diseases:

| Disease | Deficient enzyme | Main findings |
|---------|-------------------|---------------|
| Von Gierke (type I) | Glucose-6-phosphatase | Severe fasting hypoglycemia, hepatomegaly |
| Pompe (type II) | Lysosomal alpha-glucosidase | Cardiomegaly, muscle weakness |
| Cori (type III) | Debranching enzyme | Mild hypoglycemia, short outer branches |
| McArdle (type V) | Muscle glycogen phosphorylase | Exercise intolerance, muscle cramps |

The pentose phosphate pathway (PPP) - also called the hexose monophosphate shunt - runs parallel to glycolysis. It branches off at the first glycolysis intermediate (glucose-6-phosphate) and diverts it away from ATP production toward two different goals: generating NADPH and producing ribose-5-phosphate. The PPP does NOT feed the TCA cycle or ETC directly; it is a side path that produces biosynthetic precursors while glycolysis continues toward pyruvate.

Why PPP Matters

• NADPH: needed for fatty acid synthesis, cholesterol synthesis, nucleotide biosynthesis, and regeneration of glutathione (the main cellular antioxidant).
• Ribose-5-phosphate: the sugar backbone for nucleotides (DNA, RNA, ATP, NAD+, FAD, CoA).

Both products are critical. The PPP can produce just NADPH, just ribose-5-P, or both, depending on cellular needs.

Two Phases

Oxidative Phase (Irreversible)

1. Glucose-6-P → 6-phosphogluconolactone + NADPH. Enzyme: glucose-6-phosphate dehydrogenase (G6PD). Rate-limiting step.
2. 6-phosphogluconolactone → 6-phosphogluconate.
3. 6-phosphogluconate → ribulose-5-phosphate + CO2 + NADPH.

Net: 1 G6P → ribulose-5-P + 2 NADPH + CO2.

Non-Oxidative Phase (Reversible)

Ribulose-5-P can be isomerized to ribose-5-P (for nucleotides) or transketolase/transaldolase enzymes can rearrange sugars to produce glycolytic intermediates (F6P, G3P) if ribose is not needed.

G6PD Deficiency

G6PD is X-linked. Deficiency is the most common human enzyme deficiency worldwide. Without G6PD, the oxidative phase of the PPP is impaired, reducing NADPH production. This is a problem for red blood cells because:

• RBCs have no nucleus (cannot make new enzymes to replace damaged ones).
• RBCs are exposed to oxidative stress in blood.
• RBCs depend on NADPH to regenerate reduced glutathione, which detoxifies reactive oxygen species.

Without NADPH, oxidative damage accumulates, hemoglobin precipitates (forming Heinz bodies), and RBCs lyse. This causes hemolytic anemia. Triggers include fava beans, sulfa drugs, antimalarials (primaquine), and infections.

Whole-body metabolism is coordinated across organs. Two classic inter-organ cycles - the Cori cycle and the glucose-alanine cycle - redistribute carbon and nitrogen between working muscle and the liver.

Both cycles are clever ways of linking anaerobic glycolysis (fast ATP, produces lactate) in one tissue with gluconeogenesis (makes glucose from non-carb sources) in another. They show how glycolysis and gluconeogenesis - seemingly opposite pathways from sections 9.2-9.6 - work as a team across the whole body during exercise or fasting.

The Cori Cycle

Muscle at high workload (sprinting) produces lactate via anaerobic glycolysis. Lactate travels through blood to the liver. The liver converts lactate → pyruvate (by lactate dehydrogenase) and runs gluconeogenesis to make glucose, which is released into blood and can return to muscle.

Net energy transfer: muscle gains 2 ATP per glucose (glycolysis). Liver spends 6 ATP per glucose (gluconeogenesis). The muscle “borrows” energy from the liver during intense work - the liver pays the bill.

The Glucose-Alanine Cycle

A parallel cycle that transports nitrogen from muscle to liver. During exercise and fasting, muscle protein is broken down for energy. Amino acid carbons are burned locally, but nitrogen (as NH3) is toxic. Muscle transaminates pyruvate with NH3 to form alanine, which travels through blood to the liver.

In the liver, alanine is transaminated back, releasing NH3 (entering the urea cycle) and producing pyruvate (entering gluconeogenesis to make glucose). The glucose returns to muscle. So the cycle transports BOTH nitrogen (safely to the urea cycle) AND carbon (to make glucose).