Team:BGU Israel/Project/Artificial Exercise

One of the major symptoms of the metabolic syndrome is fatty liver – an abnormal accumulation of fat in the liver, whether it comes from our diet or de novo lipogenesis in the body. In fact, this phenomenon is highly correlated with all the components of the metabolic syndrome – high plasma triglycerides, hyperglycemia and insulin resistance. It may also increase the risk for type 2 diabetes and cardiovascular diseases (Bugianesi, Moscatiello,Ciaravella&Marchesini, 2010).

In order to cope with the large accumulation of fat in the liver, we looked for a remedy that will enable the body to reach high energy expenditure, thus leading to high rate of fat degradation. In our search, we learnt about the use of DNP (2,4 – dinitrophenol) as a dieting aid, in early 1930.

DNP increases the permeability of the inner mitochondrial membrane to protons, allowing them to move from the inner membrane space to the mitochondrial matrix, bypassing ATP synthase. This process weakens the coupling of the electron transport chain to the production of ATP. In an attempt to raise the pH gradient, which is negatively affected by DNP, a fast oxidation of substrates occurs while the synthesis rate of ATP stays low and oxidation energy is released as heat instead. While DNP was very effective for losing weight, it has a dangerous side effect - the heat released during the uncoupling process can cause fatal hyperthermia. The use of DNP caused numerous amounts of deaths from fever symptoms, and eventually the production of the drug was prohibited but it is still illegally used with a death toll every year.

Our goal in this project was to design a treatment that has the advantages of DNP as uncoupling factor, but with an ability to control the dangerous overheating. As DNP is a chemical molecule and as such it is very difficult to control its effect once it is in the body, we looked for a protein (which is easier to produce and control from within) that works in a similar way to that of DNP, and so we found UCP1.

Uncoupling protein 1 (UCP1) is normally found in the mitochondria of brown adipose tissue, and is used to generate heat. Its mechanism of action is very similar to that of DNP – it also works by uncoupling (as its name might suggest) the two processes needed to create ATP, but since UCP1 is a protein and not a chemical compound we had the ability to control its function and expression to our benefit. If we could make this process occur directly in the liver, where UCP1 is not naturally present, it could prove to be very useful for the fast degradation of unnecessary fat. The similarity between UCP1 and DNP also means that the end result of overheating will happen in both cases. So how could we prevent it from happening?

For that purpose, we used HSP70 promoter. HSP70 (heat shock protein) protects cells from deleterious stresses. Its expression is induced by various physiological stresses, including exposure to heat shock. The HSP70 promoter contains at least two regulatory domains; the distal domain has been shown to be responsive to heat shock (Wu, Kingston & Morimoto, 1986). The activity of the HSP70 promoter can be induced by moderate hyperthermia, reaching a noticeable function levels at around 39°C. We planned a genetic circuit with HSP70 promoter and a downstream repressor that will inhibit the synthesis ofUCP1.

As depicted in figure 1, the heat produced due to the activity of UCP1 will cause temperature to rise, activating HSP70 and inducing the synthesis of a repressor. This repressor will inhibit the synthesis of UCP1, and is its concentration in the mitochondrial membrane will be decreased due to natural protein degradation, As a result, the temperature will go down and the process could start again without reaching an endangering temperature.

Click on the picture to check out the machanism

We used TMRM (tetramethylrhodamine methyl ester) to assess the ability of UCP1 to collapse membrane potential. Membrane potential-driven accumulation of TMRM within the inner membrane region of healthy functioning mitochondria results in a dramatic increase in TMRM-associated orange fluorescence. When the mitochondrial membrane potential collapses TMRM is dispersed throughout the cell cytosol at a concentration that yields minimal fluorescence upon excitation in the optimal wavelength region.

The left series of pictures shows HepG2 cells treated with TMRM of different concentrations (left – white light, right – fluorescent), and the right series shows HepG2 previously transfected with pcDNA3.1 UCP1, also treated with TMRM of different concentrations.

There appears to be no visible difference between cells transfected with pcDNA3.1 UCP1 and the normal cell line. Also there was no difference between the different concentrations of TMRM, so next time we can use the lowest one (10 nM) or perhaps even lower.

Conclusions:

Using fluorescent microscopy with TMRM is a semi-quantitative method for the assessment of mitochondrial membrane potential. It could be that even if UCP1 was correctly expressed, it didn’t lower the membrane potential enough for us to see. Next time we could use spectrometry to more quantitatively assess the function of UCP1. Additionally, many times fatty acids are used for the activation of UCP1. We didn’t use fatty acids this time, and it might be the reason for the inactivation of UCP1.