# Lecture 17: Pentose Phosphate Pathway and Gluconeogenesis

## Biochemistry

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## Learning Objectives

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

1. Describe the oxidative and non-oxidative phases of the pentose phosphate pathway
2. Explain the importance of NADPH and ribose-5-phosphate production
3. Describe the clinical significance of glucose-6-phosphate dehydrogenase deficiency
4. List the reactions of gluconeogenesis that bypass the irreversible steps of glycolysis
5. Explain the reciprocal regulation of glycolysis and gluconeogenesis
6. Describe the Cori cycle and its physiological significance

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## Lecture Content

### I. The Pentose Phosphate Pathway (PPP) -- Overview

The pentose phosphate pathway, also called the hexose monophosphate (HMP) shunt or phosphogluconate pathway, operates in the **cytoplasm** and produces two main products: **NADPH** (reducing power for biosynthesis and antioxidant defense) and **ribose-5-phosphate** (a precursor for nucleotide synthesis, including DNA, RNA, ATP, CoA, NAD+, and FAD). The pathway does not produce ATP directly and is particularly active in the liver, adipose tissue, adrenal cortex, red blood cells, and lactating mammary gland.

### II. Oxidative Phase (Irreversible)

The oxidative phase consists of three reactions. **Glucose-6-phosphate dehydrogenase (G6PD)** catalyzes the **rate-limiting step**, oxidizing glucose-6-phosphate to 6-phosphoglucono-delta-lactone while producing the first NADPH. This enzyme is regulated by the [NADP+]/[NADPH] ratio: NADPH inhibits while NADP+ activates (when NADPH is consumed, rising NADP+ stimulates the pathway). **Lactonase** then hydrolyzes the lactone to 6-phosphogluconate. Finally, **6-phosphogluconate dehydrogenase** performs an oxidative decarboxylation, yielding ribulose-5-phosphate, CO2, and the second NADPH. The net yield of the oxidative phase per glucose-6-phosphate is 2 NADPH plus ribulose-5-phosphate plus CO2.

### III. Non-Oxidative Phase (Reversible)

The non-oxidative phase interconverts sugars of various lengths (3C, 4C, 5C, 6C, 7C) through the action of several enzymes. **Ribulose-5-phosphate isomerase** converts ribulose-5-P to ribose-5-P. **Ribulose-5-phosphate epimerase** converts ribulose-5-P to xylulose-5-P. **Transketolase** (requiring TPP/thiamine) transfers 2-carbon units, while **transaldolase** transfers 3-carbon units. This phase connects the PPP to glycolysis, as its products include fructose-6-phosphate and glyceraldehyde-3-phosphate.

The cell can adjust the ratio of NADPH to ribose-5-phosphate production through four operational modes. When ribose-5-P is needed more than NADPH, the non-oxidative phase runs in reverse from glycolytic intermediates. When both are needed equally, the oxidative phase produces both. When NADPH is needed but ribose-5-P is not, ribose-5-P is recycled back to glucose-6-P. When both NADPH and ATP are needed, ribose-5-P is converted to glycolytic intermediates for further oxidation.

### IV. Importance of NADPH

NADPH serves as the electron donor for **reductive biosynthesis** of fatty acids, cholesterol, and steroid hormones. It is critical for **antioxidant defense** through the glutathione system: glutathione reductase uses NADPH to regenerate reduced glutathione (GSH), and glutathione peroxidase uses GSH to neutralize reactive oxygen species like hydrogen peroxide. NADPH also powers the **cytochrome P450 system** for drug and xenobiotic metabolism in the liver, the **respiratory burst** in phagocytes (where NADPH oxidase produces superoxide to kill pathogens), and **nitric oxide synthesis** by NO synthase.

### V. G6PD Deficiency

G6PD deficiency is the most common human enzyme deficiency, affecting approximately 400 million people worldwide and inherited in an X-linked recessive pattern. Red blood cells are particularly vulnerable because they lack mitochondria and nuclei, making the PPP their only source of NADPH, and they face high levels of oxidative stress from oxygen transport. Without sufficient NADPH, reduced glutathione cannot be maintained. **Oxidative stress** from infections, certain drugs, or fava beans depletes GSH, leading to oxidative damage to hemoglobin and the red cell membrane and resulting in **hemolytic anemia**. Common triggers include primaquine (antimalarial), sulfonamides, dapsone, nitrofurantoin, fava beans (favism), and infections. Blood smears show **Heinz bodies** (denatured hemoglobin precipitates) and **bite cells**. The deficiency is protective against malaria (Plasmodium falciparum), explaining its high prevalence in malaria-endemic regions.

<image>A diagram of the pentose phosphate pathway. Panel A: The oxidative phase showing the three reactions from glucose-6-phosphate to ribulose-5-phosphate, with 2 NADPH and 1 CO2 produced. G6PD is highlighted as the rate-limiting enzyme. Panel B: The non-oxidative phase showing the interconversion of 3C, 4C, 5C, 6C, and 7C sugar phosphates by transketolase and transaldolase, with connections to glycolytic intermediates (fructose-6-phosphate and glyceraldehyde-3-phosphate). Panel C: The role of NADPH in maintaining reduced glutathione (GSH) and the consequences of G6PD deficiency — showing the chain from low NADPH to low GSH to oxidative damage to hemolysis, with images of Heinz bodies on a blood smear.</image>

### VI. Gluconeogenesis -- Overview

Gluconeogenesis is the synthesis of glucose from non-carbohydrate precursors. It occurs primarily in the **liver** (approximately 90%) and **kidney** (approximately 10%) and is active during **fasting, starvation, and prolonged exercise**. The precursors include **lactate** (from anaerobic glycolysis in muscle and RBCs via the Cori cycle), **glycerol** (from lipolysis of triglycerides in adipose tissue), **glucogenic amino acids** (especially alanine via the glucose-alanine cycle), and **propionyl-CoA** (from odd-chain fatty acid oxidation, converted to succinyl-CoA and then to OAA). Importantly, acetyl-CoA cannot be net converted to glucose in animals because the two carbons entering the TCA cycle as acetyl-CoA are lost as CO2 before OAA is regenerated.

### VII. Gluconeogenesis Reactions

Seven of the ten glycolytic steps are reversible and shared with gluconeogenesis. The three irreversible glycolytic steps must be bypassed.

**Bypass 1 (Pyruvate to PEP)** requires two steps. **Pyruvate carboxylase** in the mitochondrial matrix converts pyruvate to oxaloacetate using CO2, ATP, and biotin, and is activated by acetyl-CoA. **PEP carboxykinase (PEPCK)** then converts OAA to PEP using GTP. Because OAA cannot cross the inner mitochondrial membrane directly, it is transported as malate or aspartate. The net cost is 1 ATP + 1 GTP per pyruvate converted to PEP.

**Bypass 2** uses **fructose-1,6-bisphosphatase (FBPase-1)** to hydrolyze fructose-1,6-bisphosphate to fructose-6-phosphate. This enzyme is inhibited by AMP and F-2,6-BP (the same regulators that activate PFK-1) and activated by citrate and ATP.

**Bypass 3** uses **glucose-6-phosphatase**, located in the ER membrane of liver and kidney cells only, to hydrolyze glucose-6-phosphate to free glucose. Because muscle and brain lack this enzyme, they cannot release free glucose into the blood.

### VIII. Energy Cost of Gluconeogenesis

Converting 2 pyruvate to 1 glucose requires 4 ATP + 2 GTP + 2 NADH, compared to the glycolytic yield of only 2 ATP + 2 NADH per glucose. Gluconeogenesis is therefore more energetically costly than glycolysis is productive, but it is thermodynamically favorable when precursors and energy are available.

### IX. Reciprocal Regulation of Glycolysis and Gluconeogenesis

Glycolysis and gluconeogenesis are coordinately regulated to prevent futile cycling. **Fructose-2,6-bisphosphate (F-2,6-BP)** is the key reciprocal regulator, activating PFK-1 (glycolysis) and inhibiting FBPase-1 (gluconeogenesis). During fasting, glucagon increases cAMP, activating PKA, which phosphorylates PFK-2/FBPase-2 to activate its FBPase-2 activity, lowering F-2,6-BP and thereby slowing glycolysis while accelerating gluconeogenesis. In the fed state, insulin promotes dephosphorylation, activating PFK-2, raising F-2,6-BP, and favoring glycolysis. Additional reciprocal regulators include AMP (activates PFK-1, inhibits FBPase-1), citrate (inhibits PFK-1, activates FBPase-1), and acetyl-CoA (activates pyruvate carboxylase for gluconeogenesis while inhibiting PDC).

### X. The Cori Cycle

In the Cori cycle, lactate produced by anaerobic glycolysis in muscle and red blood cells is transported via the blood to the liver, which converts it back to glucose through gluconeogenesis. The glucose is then released back into the blood for use by peripheral tissues. The net effect shifts the energy burden to the liver: it costs 6 ATP in the liver to regenerate one glucose that yields only 2 ATP in the muscle, for a net cost of 4 ATP paid by the liver.

<image>A diagram showing the reciprocal regulation of glycolysis and gluconeogenesis in the liver. The left side shows glycolysis enzymes (hexokinase/glucokinase, PFK-1, pyruvate kinase) with their activators and inhibitors. The right side shows the gluconeogenic bypass enzymes (glucose-6-phosphatase, FBPase-1, pyruvate carboxylase/PEPCK) with their regulators. In the center, fructose-2,6-bisphosphate is shown as the key reciprocal regulator with PFK-2/FBPase-2 bifunctional enzyme depicted above it, showing how glucagon (fasting) and insulin (fed) control its activity through phosphorylation. The Cori cycle is shown as an inset: lactate flows from muscle to liver, glucose flows from liver to muscle.</image>

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