Everted gut sacs: why an ATPase inhibitor blocks glucose absorption
Practical 4: glucose uptake in the jejunum. Students use everted sacs of rat jejunum (each 3 cm long, about 0.20 g wet mass) filled with 1 mL of buffer and bathed in 25 mL of oxygenated buffer at 37 °C. The bathing solution contains 5 mmol/L glucose and 140 mmol/L Na⁺. After 20 min the glucose concentration inside each sac is measured, and uptake is expressed as µmol glucose per gram wet mass. Four conditions were run in triplicate:
- Control: 41 µmol g⁻¹ (SD 3)
- 0.1 mmol/L phlorizin: 4 µmol g⁻¹ (SD 1)
- 1 mmol/L ouabain, added 10 min before glucose: 9 µmol g⁻¹ (SD 2)
- low Na⁺ (25 mmol/L, choline chloride replacing NaCl): 12 µmol g⁻¹ (SD 2)
The class had predicted that ouabain would have no effect, because ouabain does not bind the glucose carrier. In a separate run, 5 mmol/L fructose in the presence of 0.1 mmol/L phlorizin gave 38 µmol g⁻¹.
1. Explain the ouabain result. Refer explicitly to the type of transport involved and to the energy source that ultimately drives glucose accumulation.
Read the worked answer
Ouabain never touches SGLT1, yet uptake falls from 41 to 9 µmol g⁻¹, an inhibition of about 78 %. The reasoning chain runs through the energy supply, not the carrier:
- SGLT1 is a secondary active transporter. It moves glucose against its concentration gradient by coupling to Na⁺ moving down its electrochemical gradient (2 Na⁺ per glucose).
- That Na⁺ gradient is not self-sustaining. It is created and maintained by the basolateral Na⁺/K⁺-ATPase, which exports 3 Na⁺ and imports 2 K⁺ per ATP hydrolysed.
- Ouabain blocks the ATPase. Na⁺ that leaks into the enterocyte is no longer pumped out, so cytosolic [Na⁺] rises and the inward Na⁺ gradient collapses.
- With the driving force gone, glucose can no longer be accumulated. Ouabain is therefore an indirect inhibitor: glucose absorption is ATP-dependent even though SGLT1 itself hydrolyses no ATP.
The residual 9 µmol g⁻¹ (above the 4 µmol g⁻¹ seen with phlorizin) is consistent with a gradient that decays during the 20 min rather than disappearing instantly, plus a small Na⁺-independent component. A useful control would be to add ouabain together with phlorizin: if uptake then equals the phlorizin value, the residual flux is carrier-independent.
2. Evaluate whether the low-Na⁺ result is consistent with a purely Na⁺-coupled model. What does the gap between 12 and 4 µmol g⁻¹ suggest, and how could you test your suggestion?
Read the worked answer
A purely Na⁺-coupled model does not predict zero uptake at 25 mmol/L external Na⁺. Cytosolic Na⁺ in enterocytes is roughly 10–20 mmol/L, so at 25 mmol/L outside there is still a (smaller) inward gradient and SGLT1 still has a driving force. A lower but non-zero rate is exactly what the data show, so the low-Na⁺ result is qualitatively consistent with Na⁺ coupling.
The comparison that needs explaining is 12 versus 4 µmol g⁻¹. Phlorizin removes the carrier itself; low Na⁺ only weakens its driving force. Two conditions are therefore not equivalent, and the ~8 µmol g⁻¹ excess over the phlorizin value indicates a Na⁺-independent route. Candidates: paracellular leakage through tight junctions, and basolateral GLUT2 re-entry of glucose that entered by another path. At only 5 mmol/L luminal glucose, apical GLUT2 recruitment is expected to be weak, so the paracellular explanation is the more economical one.
Testable predictions: (a) combining low Na⁺ and phlorizin should reduce uptake to about the phlorizin value if the extra flux is carrier-independent; (b) adding a tight-junction opener should raise the low-Na⁺ value but not the phlorizin value; (c) an SGLT1 knockdown should abolish the Na⁺-dependent fraction only. A good answer states one hypothesis, one prediction and one control rather than listing possibilities.
3. Fructose uptake (38 µmol g⁻¹) is barely affected by phlorizin. Explain this and predict, with justification, whether 1 mmol/L ouabain would reduce fructose uptake in a 60-minute experiment.
Read the worked answer
Fructose enters via GLUT5, a facilitated diffusion carrier that is not coupled to Na⁺ and has no phlorizin-binding site. Phlorizin therefore cannot inhibit it, and the near-identical value (38 vs 41 µmol g⁻¹) is the expected result. This is the internal control that proves phlorizin's effect on glucose is specific to SGLT1 rather than a general poisoning of the tissue.
For ouabain the prediction is less certain and should be stated as such. Two opposed considerations:
- Against an effect: fructose flux does not require the Na⁺ gradient; GLUT5 is driven only by the fructose gradient, so collapsing the Na⁺ gradient should not directly change it.
- For an effect at 60 min: ouabain poisoning of the ATPase is expected to lower cellular ATP, cause Na⁺ loading, cell swelling and eventually loss of viability. Over an hour this would reduce any transport process, including facilitated diffusion, and could also disturb apical GLUT2 trafficking. A partial reduction, not abolition, is the most likely outcome.
The decisive control is a viability/ATP assay at 60 min (or a ouabain + phlorizin + fructose run). Without it, a fall in fructose uptake could not be attributed to loss of the Na⁺ gradient. This is the judgement the question rewards: distinguishing a mechanism-specific prediction from a general toxicity effect.