Glycolysis and Gluconeogenesis

Glycolysis—The 10-Step Pathway

• Step 1 (Glucose → Glucose-6-phosphate): Hexokinase or glucokinase catalyzes phosphorylation; hexokinase is inhibited by G6P (feedback inhibition) while glucokinase is not, allowing hepatic glucose sensing; this step is irreversible and traps glucose in the cell
• Steps 2-4 (Isomerization and phosphorylation): Phosphoglucose isomerase, phosphofructokinase (PFK), and aldolase convert G6P to DHAP and G3P; this phase consumes 2 ATP
• Step 3 (G6P → F1,6BP via PFK): Rate-limiting enzyme of glycolysis; inhibited by ATP, citrate, and fatty acids; activated by AMP, ADP, and F2,6BP (primary allosteric regulator); this is the major control point of the pathway
• Steps 5-9 (Oxidation and phosphorylation): Glyceraldehyde-3-phosphate dehydrogenase oxidizes G3P using NAD+ to form NADH; subsequent phosphorylation generates 1,3-bisphosphoglycerate, which transfers its phosphate to ADP (substrate-level phosphorylation); net production of 2 ATP and 2 NADH per glucose
• Step 10 (Pyruvate production): Pyruvate kinase catalyzes PEP → pyruvate, generating ATP; inhibited by ATP and alanine; activated by F1,6BP (feedforward activation); this is a key control point

Gluconeogenesis—The Reverse Pathway

• Three irreversible steps bypass: Pyruvate carboxylase (pyruvate → oxaloacetate in mitochondria; requires biotin and ATP), PEPCK (oxaloacetate → PEP in cytoplasm; rate-limiting enzyme; requires GTP), and fructose-1,6-bisphosphatase (F1,6BP → F6P; inhibited by F2,6BP and AMP)
• Substrate sources: Pyruvate (from lactate via Cori cycle, from amino acids, from glycerol-3-phosphate), alanine (via glucose-alanine cycle), and glycerol (from triglyceride breakdown); fatty acids cannot serve as net gluconeogenic substrates (acetyl-CoA cannot be converted to oxaloacetate)
• Location: Primarily hepatic (90%) and renal (10%); kidney becomes increasingly important during prolonged fasting; adipose and muscle lack glucose-6-phosphatase, preventing glucose release
• Allosteric regulation: F2,6BP is the master switch—high F2,6BP (fed state) activates PFK and inhibits F1,6BP-ase, favoring glycolysis; low F2,6BP (fasted state) promotes gluconeogenesis; glucagon decreases F2,6BP via inhibition of PFK-2
• Hormonal control: Glucagon and cortisol increase gluconeogenesis; insulin suppresses it by decreasing substrate availability and allosteric inhibition; epinephrine's effects depend on tissue and alpha vs. beta signaling

Hyperglycemia (from glycolysis dysfunction or gluconeogenic excess)

• Polyuria, polydipsia, and weight loss (osmotic diuresis from hyperglycemia)
• Fatigue and weakness from cellular glucose uptake impairment
• Recurrent infections due to impaired immune function and hyperglycemic environment
• Visual disturbances (cataracts from osmotic stress; retinopathy from microvascular disease)

Hypoglycemia (from glycolytic insufficiency or gluconeogenic failure)

• Autonomic symptoms: tremor, palpitations, diaphoresis, anxiety (from catecholamine release)
• Neuroglycopenic symptoms: confusion, altered behavior, seizures, loss of consciousness, focal neurologic deficits
• Infants with glycogen storage diseases present with hepatomegaly, failure to thrive, and seizures from severe fasting hypoglycemia
• Lactic acidosis may accompany hypoglycemia in certain enzyme deficiencies (e.g., pyruvate dehydrogenase deficiency)

Exercise and Starvation States

• During intense exercise: initial ATP depletion activates glycolysis; lactate accumulation (Pasteur effect); hyperventilation if severe
• Prolonged fasting: ketone breath odor, weight loss, weakness, confusion if prolonged, and eventual starvation ketoacidosis

Clinical pearls

• Neonates are particularly vulnerable to hypoglycemia because hepatic glycogen is limited and they cannot yet mobilize fat effectively
• Glucose-6-phosphatase deficiency presents as severe hypoglycemia on fasting with massive hepatomegaly but no lactic acidosis (G6P cannot be further metabolized)—distinguished from other glycogenoses by the virtual absence of glucose output

Laboratory evaluation of glycolytic/gluconeogenic disorders

• Fasting glucose, lactate, and pyruvate: Hypoglycemia with elevated lactate suggests pyruvate dehydrogenase deficiency or pyruvate carboxylase deficiency; isolated hypoglycemia with normal lactate suggests G6P-ase deficiency or PEPCK deficiency
• Glucose response to glucagon stimulation: Normal response rules out severe hepatic glycogenolysis defects; absent or blunted response indicates gluconeogenic enzyme deficiency
• Enzyme assay in liver or muscle biopsy: Definitive diagnosis; required for glycogen storage diseases (e.g., acid maltase deficiency = GSD II; phosphorylase deficiency = GSD V)
• Genetic testing: Increasingly available for known mutations in GCK, PYGL, G6PC, and other genes
• Metabolite profiling: Elevated alanine suggests gluconeogenic block; elevated lactate suggests oxidative enzyme defects
• Liver ultrasound: Assess for hepatomegaly (common in glycogen storage diseases)
• Oral glucose tolerance test (OGTT): Elevated 2-hour glucose and normal fasting glucose may indicate impaired glycolytic capacity; poor gluconeogenic suppression seen in type 2 diabetes

Management of Hypoglycemia (acute)

• First-line: Glucose administration (15-20 g rapidly absorbed carbohydrate orally if conscious; examples: 4 oz juice, 6-8 glucose tablets, 1 tbsp honey); recheck glucose in 15 minutes and repeat if needed
• For severe or unconscious patients: IV dextrose 0.5-1 g/kg (typically 25 mL of 50% dextrose) or glucagon 1 mg IM/IV/SC (raises blood glucose via increased glycogenolysis and gluconeogenesis over 10-20 minutes)
• Maintenance: Long-acting carbohydrates (bread, milk) to prevent recurrence

Management of Chronic Glycolytic/Gluconeogenic Disorders

• Frequent meals (every 2-3 hours) with complex carbohydrates to maintain blood glucose and reduce metabolic stress
• Uncooked cornstarch (1-2 g/kg every 4-6 hours, especially bedtime) provides slow glucose release in glycogen storage diseases
• Avoid fasting: Nocturnal feeding tube may be necessary in severe disease
• Allopurinol for gout prevention (uric acid overproduction in glycogenoses)
• Avoid high-fat diets in some glycogenoses

Enzyme facts examiners return to

• Hexokinase vs. glucokinase: hexokinase is ubiquitous with low K_m/low V_max and is feedback-inhibited by G6P; glucokinase (liver, pancreatic β cell) has high K_m/high V_max, is insulin-induced, and acts as the glucose sensor. Heterozygous GCK loss-of-function causes MODY: mild, stable fasting hyperglycemia in a lean adolescent with a strong autosomal dominant family history and no autoantibodies — the classic distractor is calling it type 1 diabetes.
• Fructose-2,6-bisphosphate is the answer whenever a stem asks how glucagon flips the liver from glycolysis to gluconeogenesis: PKA phosphorylates the bifunctional PFK-2/FBPase-2 enzyme, lowering F2,6BP, which de-activates PFK-1 and relieves inhibition of fructose-1,6-bisphosphatase.
• Pyruvate carboxylase requires biotin (as do acetyl-CoA carboxylase and propionyl-CoA carboxylase) and is allosterically activated by acetyl-CoA — the signal that fat oxidation is under way and pyruvate should go to oxaloacetate rather than acetyl-CoA.
• Odd-chain fatty acids are gluconeogenic via propionyl-CoA → succinyl-CoA; even-chain fatty acids are not. Consistent with the framing above, acetyl-CoA cannot yield net glucose.

Toxin and drug associations

• Arsenic inhibits glyceraldehyde-3-phosphate dehydrogenase (and lipoic acid–dependent PDH); look for garlic breath, rice-water stools, QT prolongation. Fluoride inhibits enolase — hence the gray-top glycolysis-inhibitor tube.
• Ethanol: cytosolic NADH excess drives pyruvate → lactate and oxaloacetate → malate, blocking gluconeogenesis; the binge-drinking, poorly fed patient presents with fasting hypoglycemia plus lactic acidosis.
• Metformin lowers hepatic gluconeogenic output and is first-line pharmacotherapy for type 2 diabetes per the ADA Standards of Care; the tested adverse effect is lactic acidosis, and ADA advises against initiation at markedly reduced eGFR.

Deficiencies with a signature phenotype

• Pyruvate kinase deficiency: RBCs (no mitochondria) depend entirely on glycolysis — chronic hemolytic anemia with rising 2,3-BPG and a right-shifted O₂ curve; second most common enzymatic cause of hemolysis after G6PD deficiency.
• Aldolase B deficiency (hereditary fructose intolerance): fructose-1-phosphate traps inorganic phosphate, arresting both glycogenolysis and gluconeogenesis — vomiting and hypoglycemia when juice or sucrose is introduced; treatment is dietary exclusion of fructose, sucrose, and sorbitol.
• Best next step in unexplained hypoglycemia: obtain the critical sample (glucose, insulin, C-peptide, proinsulin, β-hydroxybutyrate, sulfonylurea screen) during the spontaneous episode before giving dextrose, per Endocrine Society guidance on hypoglycemic disorders.