Question

Some scientists believe that fructose is particularly bad for human health because it promote fat synthesis, contributing to development of type II diabetes and fatty liver disease. You are a scientist working for a lab attempting to develop a drug to help with such diseases called fructono. The idea is that fructono could be added to the diet to reduce fructose absorption; even though fructono would be absorbed, it would not be metabolized, and would be excreted by the kidney. The hypothesis is that fructono binds to the fructose transporter, competitively displacing fructose. This cannot be easily studied in humans, so you are investigating this hypothesis with a mouse small intestine, which is known to transport fructose using a transporter that is quite similar in amino acid sequence and structure to the human protein.

For your research, you set up a perfused intestine prep. You cut out about 1 cm of small intestine, and put it in a dish containing saline. You insert a tube into the intestinal section, and use a pump to push any fluid you want through the lumen of the intestine. You have radio-labeled fructono, so you can measure its appearance in the saline outside the intestine.

How can you measure the rate of transport of fructono?

Describe experiments that would test whether the absorption of fructono is protein-mediated vs. passive (just leaking through cracks, or diffusing through membranes). What results will you get if the transport is protein-mediated vs. passive?

Describe experiments that would test whether the absorption of fructono is active, and the predicted results if transport is active or not.

Describe experiments that will test whether the active transport of fructono requires luminal Na+, and the predicted result if transport is Na+-dependent or not.

Describe experiments that will test whether fructono can reduce the transport of fructose in a dose-dependent manner.

What side-effects might fructono have?

Answer 1

Fructose enters through facilitated diffusion. It is a process of pasive transport of molecules across membrane mediated by specific transmembrane integral proteins. GLUT5 is the fructose transporter present on the membrane.

The transport of fructose can be measured by filtration technique. Luminal perfussion model and Western blotting can measure the activity of GLUT5 proteins. Patch-clamp technique can be used to study the single transport protein to test whether it is active or not.

Fructose are transported only with the help of Na+. So we can test the rate by doing an experiment by adding Na+ along with fructono in a test fluid from intestine and provide a control for the experiment i.e., without adding Na+ (make sure to add equal concentration of fructono to each set up). As fructono is a substitute of fructose, when it competitively binds with GLUT5, the initial and final concentration of it can be monitored by spectrophotometer and thus we can analyse.

Then for understanding the competition between fructono and fructose the same experiment can be repeated by adding one more i.e., fructose+fructono+Na+test fluid in appropriate concentration and it's control i.e., without adding fructono to the same volume. Then the activity can be analysed spectrophotometerically or u can do it in microplate reader using any dye like azocaesin. We can do this by taking different concentration to understand in which concentration the activity is showing high.

Side-effects of fructono - as it displaces fructose uptake, the fructose malabsorption can cause abdominal pain, nausea, acidity etc.