Question

You are a scientist working for a lab attempting to develop an anti-obesity drug called glucono. The idea is that glucono could be added to the diet to reduce glucose and calorie absorption; even though glucono would be absorbed, it would not be metabolized, and would be excreted by the kidney. The hypothesis is that glucono binds to the Na+/glucose transporter that is a part of secondary active transport of glucose. This cannot be easily studied in humans, so you are investigating this hypothesis with a mouse small intestine, which is known to transport glucose by secondary active transport, 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 glucono, so you can measure its appearance in the saline outside the intestine

1) Describe three experiments that will test whether the absorption of glucono is active (protein-mediated and energy-requiring) vs. passive (e.g. just leaking through cracks, or diffusing through membranes). What results will you get if the transport is active vs. passive?

2) Describe one experiment that will test whether the active transport of glucono requires luminal Na+. 2 pts.

3) Describe one experiment that will test whether glucono can reduce secondary active transport of glucose in a dose-dependent manner. 2 pts

4) List two possible side-effects of glucono. 2 pts

Answer 1

There are two main ways a molecule can enter a cell, they can be diffused or they can be transported. Diffusion occurs in a gradient, this means that molecules will flow from where there are more molecules to where there is less, this is normally a slow process but it doesn’t cost energy. If the molecule being diffused is water, it is called osmosis. Transport is a faster method and it is mediated by a protein that crossed the cell membrane. There are two types of transports, which are passive and active. Passive transports do not require energy but must follow the gradient and active transporters cost energy to use but can go against a molecules gradient. Passive transport can also be called facilitated diffusion , as it continuous diffusion movement but faster. The energy required to use active transport comes from ATP, a high density energy molecule, or from an ionic gradient, as electric energy.

The Na/glucose transporter uses the natural gradient casued by the Na/K bomb to incorporate glucose into the cells, particularly in epithelial tissues. So for experiment 1 you could use a solution with Cl^- and glucono. In this experiment you would expect glucono not to be absorbed by the cells as the Na⁺ gradient would be broken by the formation of NaCl salt. If it is passive the glucono would still be incorporated.

For experiment 2 you can deprive your tissue of ATP. Without ATP the Na/K bomb will cease to function leading to an equilibrium of the ions. If the glucono requires active transport it will not be incorporated.

For experiment 3 you can mutate the Na/glucose transporter as to make it loss function. If the glucono can be transported passively it will enter the cells.

In the three experiments you would use glucose as a positive control and you can use any other monosaccharide as a negative control. This means glucose must behave as the glucono and the other monosaccharide must not behave the same.

2) Experimetn 1 can also test if the Na⁺ is required in the lumen of the organism. As the salt NaCl is formed the Na⁺ concentration in the lumen is decreased, thus making experiment 1 can tell you two things in one experiment.

3) For this experiment you require to establish the intake rate of glucose to the tissue. This can be done by introducing a known quantity of glucose in the lumen and quantifying how much it came out of the lumen. Next you would repeat the same experiment but adding different glucono concentrations, if it dose dependent it would increase the glucose that goes out the lumen with the increase of glucono.

4) If glucono is similar to glucose it can activate the mechanism of glucose without the intake of energy. This could lead to metabolism disequilibrium such as diabetes and insulin insensitivity. This can be caused as the energetic requirement is not reached even though the signal associated with glucose is received, this can also lead to an increase of insulin in the blood generating the resistance. It can also cause a sensation of fatigue, as the organism perceives it receives energy when it does not.