Glucose uptake across an intestinal monolayer: who pays the ATP?
A line of human intestinal epithelial cells (Caco-2) is grown as a tight monolayer on a permeable filter, with the apical surface facing the upper chamber. The upper chamber contains 2 mM glucose and 140 mM Na⁺; the lower chamber contains 5 mM glucose. After 30 minutes the cells contain 30 mM glucose and the lower chamber has gained glucose.
Three experiments are then run on identical monolayers.
Experiment A: phlorizin, a competitive inhibitor of the Na⁺/glucose cotransporter SGLT1, is added to the upper chamber only. Glucose entry stops within seconds and intracellular glucose falls.
Experiment B: phloretin, which blocks the basolateral facilitated transporter GLUT2, is added to the lower chamber only. Intracellular glucose rises above 60 mM and glucose no longer appears below.
Experiment C: ouabain, which binds and inhibits the Na⁺/K⁺-ATPase, is added to the lower chamber. For the first 10 minutes uptake continues almost normally; by 25 minutes intracellular ATP has fallen to about 20% of control and net glucose uptake has almost stopped.
The cells are not damaged, and GLUT2 protein levels are unchanged in all three experiments.
1. Explain how intracellular glucose reaches 30 mM when the medium contains only 2–5 mM, and why ouabain stops uptake although SGLT1 itself does not hydrolyse ATP. Account for the 10-minute lag.
Read the worked answer
Energy source. SGLT1 does not hydrolyse ATP. It is a secondary active transporter: it couples the uphill movement of glucose (2 mM outside → 30 mM inside) to the downhill movement of Na⁺ (140 mM outside → roughly 10–15 mM inside). The Na⁺ gradient is generated by the basolateral Na⁺/K⁺-ATPase, which exports 3 Na⁺ and imports 2 K⁺ per ATP hydrolysed. The ATP is spent one step upstream, not at the glucose site.
Why ouabain works with a delay. Ouabain blocks the pump, so intracellular Na⁺ rises and the transmembrane Na⁺ gradient collapses. Once the gradient is gone, SGLT1 has no driving force and glucose entry stops. The lag is expected: the pre-existing gradient takes minutes to run down, and glucose already inside leaks back out passively through GLUT2, which is unchanged and still functional. ATP also falls to 20% because the pump is a major consumer - that does not directly affect SGLT1 (which needs no ATP) but shows the two events are separable in time.
Key inference. Uptake should stop when the Na⁺ gradient is abolished, not when ATP alone is low. Experiment C demonstrates exactly this separation: at 10 minutes uptake is normal while the pump is already inhibited, and only later, when the gradient has dissipated, does uptake fail.
2. Predict the result of replacing all apical Na⁺ with choline, a cation that SGLT1 does not transport, and justify the prediction. What controls would make the experiment interpretable?
Read the worked answer
Prediction: no accumulation. Intracellular glucose falls to roughly the external concentration (a few mM) or lower, because basolateral GLUT2 keeps removing glucose into the 5 mM lower chamber.
Reasoning: SGLT1 is an obligatory cotransporter - it cannot complete its conformational cycle without a Na⁺ ion bound and delivered to the cytoplasm. Removing Na⁺ removes the driving ion, so the coupling that produced a 30 mM internal concentration disappears. Any residual entry would have to go through apically located GLUT2, which is passive: with 2 mM outside and 30 mM inside it would mediate net efflux, not influx. So the experiment should show loss of the gradient, not merely a slower rate.
Controls:
- Choline chloride must replace NaCl at the same molarity and charge, so osmolarity and membrane potential are unchanged (a sugar substitute would change tonicity).
- Viability and ATP must be checked, to exclude toxicity of choline or of the incubation.
- Phlorizin should be added in a parallel chamber as a positive control: if any uptake survives Na⁺ removal, phlorizin distinguishes SGLT1-independent routes.
- GLUT2 protein should be measured, since Caco-2 cells can insert GLUT2 apically when glucose is high.
3. Oral rehydration solution (ORS) contains both glucose and NaCl. Using the results of these experiments, explain why adding glucose to a salty drink increases water absorption, and why a drink containing glucose but no sodium would be harmful.
Read the worked answer
Why glucose helps. SGLT1 moves 2 Na⁺ per glucose. In the gut lumen glucose pulls Na⁺ into the enterocyte, and Na⁺ uptake is followed by Cl⁻ and then by water, which moves osmotically through tight junctions and aquaporins. In secretory diarrhoea (e.g. cholera) the glucose-coupled Na⁺ route is the one absorptive pathway that still works, which is why ORS cut mortality from about 30% to under 1%. Experiment A is the pharmacological version of deleting Na⁺: block SGLT1 and the glucose–Na⁺–water coupling disappears, which shows the coupling is obligatory and not additive.
Why glucose without Na⁺ fails. Glucose is then absorbed only slowly by facilitated diffusion, without moving Na⁺; the glucose left in the lumen is an unabsorbed osmolyte that holds water in the gut and can worsen diarrhoea - the mechanism behind juice or soft drinks given to a dehydrated child.
Formulation link. The Caco-2 kinetics predict the recipe: both components, near SGLT1 stoichiometry, at concentrations high enough to saturate the transporter (Km for glucose in the low mM range) without adding an unnecessary osmotic load.