Apical glucose uptake in Caco-2 monolayers
Researchers study glucose absorption using Caco-2 monolayers grown on permeable supports. Apical and basolateral chambers both contain 5 mM glucose and 140 mM Na+. Apical uptake of 3-O-methylglucose (3-OMG), a non-metabolized glucose analog, is measured over 5 minutes and normalized to protein. Control uptake is 12.0 pmol min^-1 mg^-1. When apical Na+ is replaced by choline, uptake falls to 2.0. Adding 100 µM phlorizin to the apical side lowers uptake to 2.5. Adding 1 mM ouabain to the basolateral side lowers uptake to 3.0. Raising apical glucose from 5 mM to 25 mM increases uptake only to 13.0. The cells form tight junctions with transepithelial electrical resistance above 1000 Ω·cm². A separate assay shows that 25 mM glucose increases basolateral glucose appearance, but that increase is blocked by cytochalasin B, an inhibitor of GLUT transporters. The lab concludes that apical uptake is entirely by facilitated diffusion.
1. Evaluate the lab's conclusion that apical 3-OMG uptake is entirely by facilitated diffusion. Which observations support or contradict that conclusion, and what transport mechanism fits the data?
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Facilitated diffusion through a GLUT would be Na+-independent and insensitive to ouabain, because it uses the glucose gradient rather than the Na+ gradient. The apical uptake is strongly Na+-dependent (12.0 to 2.0 when Na+ is replaced) and phlorizin-sensitive (12.0 to 2.5). Phlorizin is a competitive inhibitor of SGLT cotransporters, not a classic GLUT inhibitor. Basolateral ouabain also lowers uptake to 3.0; ouabain inhibits the Na+/K+-ATPase, reducing the inward Na+ gradient that drives SGLT. That is a secondary-active-transport signature: apical Na+/glucose cotransport, with the Na+ gradient maintained by basolateral Na+/K+-ATPase. The residual 2.0 pmol min^-1 mg^-1 in Na+-free conditions is consistent with a smaller GLUT-mediated component, so the conclusion is partly right about a minor pathway but wrong that uptake is entirely facilitated diffusion. The tight junctions rule out a paracellular leak as the main explanation.
2. Apical glucose rising from 5 mM to 25 mM increases uptake only from 12.0 to 13.0 pmol min^-1 mg^-1. Explain this result in terms of transporter kinetics and assess the statement that more glucose always means more glucose absorption.
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A fivefold increase in substrate producing an 8% increase in rate is the signature of saturation. For a carrier with Michaelis-Menten behavior, rate = Vmax[S]/(Km + [S]). If Km is well below 5 mM, the transporter is already near Vmax at 5 mM; at 25 mM the fractional occupancy rises only slightly. This fits SGLT1, whose Km for glucose is in the sub-millimolar to low millimolar range. If apical uptake were simple facilitated diffusion with a high-Km GLUT, the 25 mM step would produce a much larger increase. The statement is therefore false over the saturating range; absorption can increase with luminal glucose only when transporters are not saturated or when additional transporters are recruited. The basolateral cytochalasin B result concerns exit from the cell, not apical entry, so it does not rescue the lab's claim. In vivo, higher luminal glucose can also increase the gradient for basolateral GLUT2, but the rate-limiting apical step still saturates.
3. Predict how removing apical Na+ would affect basolateral glucose appearance. Then design one experiment to test whether phlorizin lowers uptake by directly blocking SGLT1 or by depleting cellular ATP.
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If apical SGLT is the main entry step, removing apical Na+ should reduce intracellular glucose and therefore reduce basolateral glucose appearance; the basolateral GLUT can only export what has entered. Because 3-OMG is not metabolized, the appearance of native glucose is the relevant readout for the coupled system. To separate direct blockade from ATP depletion, measure cellular ATP under phlorizin and compare it with ouabain; if phlorizin does not lower ATP but still inhibits uptake, direct SGLT blockade is supported. A stronger design uses a membrane-impermeant phlorizin analog, or expresses SGLT1 in a cell lacking other glucose transporters and tests phlorizin with a non-metabolizable substrate. Including a positive control (ouabain, which collapses the Na+ gradient) and a negative control (cytochalasin B for GLUT) makes the result interpretable. If phlorizin lowered ATP substantially, a metabolic effect could not be excluded without these controls.