Shoelaces and Uncoupled Respiration

The world is a beautifully complex place. There is hardly ever just one way to complete a task. Take tying shoes for example: the battle still rages on whether they most effective way is utilizing the bunny ears method or just the one loop technique. Either way, the job gets done. Biochemistry is no exception to this rule. Many biological systems have more than one way to accomplish the same process. Thermogenesis, the process of the body to produce heat, has more than one mechanism for this to happen. The human body does this in efforts to maintain its internal temperature homeostasis. In their recent article, authors Jonathan Long et al. uncovers PM20D1, a new enzyme that leads to another method of undergoing thermogenesis. Before understanding how and why PM20D1 becomes involved with this process, its predecessor needs to be discussed first.

The most researched thermogenesis pathway deals with uncoupling protein 1 (UCP1). This protein is present in two types of adipose tissues, brown and beige fats2. UCP1 disrupts the natural proton gradient established by oxidative phosphorylation. The protein pumps protons back into the mitochondrial matrix without producing ATP, but producing heat in the process1. This process is known as uncoupled respiration (Fig. 1). In addition to expression of UCP1, scientists continually test the many different roles these specific adipose tissues may be involved in. The first step of this study investigates other possible pathways of undergoing uncoupled respiration. Long et al. suspect co-expression of another gene along with Ucp1. The research team proceeds to compile a list of what they refer to as “core thermogenesis”2 genes along with what genes are expressed in the same conditions as UCP1. This is how peptidase M20 domain containing 1 (PM20D1) becomes the focus of this study. With a signal peptide and no transmembrane domains, PM20D1 is seen as a secreted enzyme. To confirm this, tagged Pm20d1 gene is transfected into cells and found to be expressed in both the cell cultures as well as the in the media. With that data supporting the idea PM20D1 is a secreted enzyme, the authors move on to determining the effects of the enzyme on a live biological system and transfect mice test subjects. While on a high fat diet, the mice expressing the PM20D1 show a decrease in weight gain compared to the mice transfected with GFP as a control. The difference in weight is due to different percentage of fat mass compared to lean mass. Complementary to the weigh results, a calorimetry measurement show that mice with PM20D1 has a higher energy spending despite the same exact diet between the PM20D1 mice subjects and control GFP mice subjects. Both findings suggests that this enzyme at fault.

After seeing that PM20D1 is likely to influence biomass regulation and energy exertion, the authors now have to determine what exactly is occurring with the enzyme. With knowing that PM20D1 is part of the mammalian M20 peptidase family and how this family tends to conduct activity on small molecule substrates2, the authors use liquid chromatography mass spectrometry (LC-MS) to determine PM20D1 molecular effects in the blood. Mice injected with the PM20D1 vectors show more MS peaks with a m/z of 428, which corresponds to the molecular ion of N-oleoyl phenylalanine. Tandem mass spectroscopy supports that peak’s identity by showing a m/z peak of 164 that corresponds to a phenylalanine ion. In addition to N-oleoyl phenylalanine, there are other N-acyl amino acids found in the blood samples of the PM20D1 mice. Mass spectroscopy and its derivatives are able determine the presence of certain metabolites and help compare what is found in PM20D1 mice and the GFP mice.

With the high levels of N-acyl amino acids in PM20D1 mice compared to GFP mice, it is likely that PM20D1 is the enzyme responsible in the compounds’ formations in vivo. In vitro techniques are required to controllably examine any products that would be made, if any, with purified mouse PM20D1, fatty acids, and amino acids. LC-MS shows how N-oleoyl phenylalanine was made after using oleate and phenylalanine as reactants. The synthesis reaction uses other amino acids, apart from glutamate and ethanolamine, but the phenylalanine is the most successful amino acid. The hydrolytic reaction occurs by the enzyme breaking the N-acyl amino acid back into the fatty acid and amino acid components. By mutating PM20D1, the authors can determine which residues are essential for catalytic activity. Only the wildtype of the enzyme produces any activity. Experiments with recombinant human PM20D1 determines if the enzyme functions similarly to the mouse version. The human version displays the same conserved catalytic residues and still acts as a bidirectional enzyme.

After determining what products are created and how via PM20D1, the authors can focus on how these metabolites contribute to uncoupled respiration. After disabling the ATP synthase in adipose tissue, the addition of N-oleoyl phenylalanine increases cellular oxygen consumption, meaning uncoupled respiration is occurring. Other N-acyl amino acids also increase the cellular oxidation consumption. Experiments that utilize cells that do not express UCP1 shows that addition of N-acyl amino acids increases oxygen consumption rates, meaning that UCP1 is not needed for PM20D1 to activate uncoupled respiration. More experiments show by adding modifications to the amino group of the N-acyl amino acids halts the respiration mechanism. The amino group is necessary for the reaction to occur. Isolating the mitochondria from the cells and treating it with the N-acyl amino acids reveals  that respiration still happens, which suggests no organelles are needed to assist with uncoupled respiration.

Long et al. discussed significant findings utilizing common techniques such as LC-MS to purify and identify metabolites, western blot to determine if the enzyme of interest was present, and in vitro assays to test reaction parameters. Finding and characterizing this enzyme holds real promise for the future. When testing PM20D1 functions in vivo, mouse test subjects expressing the enzyme was shown to lose fat mass despite being on a high fat diet. The authors note that the weight loss was most likely a combination of both burning the fat and triggering a decrease in food consumption. Past studies have shown synthetic chemical uncouplers can lead to lethal side effects2. By uncovering both the enzyme and N-acyl amino acid metabolites, new targets for drug therapy has been brought to the table. Type 2 diabetes has been linked to obesity. If new therapies can be developed around PM20D1 that deal with weight loss, it could help diminish a risk factor of developing diabetes. This newly studied uncoupled respiration pathway holds potential to helping create a healthier environment. This could be a step forward in the right direction.

 

 

 

References:

  1. Brondani L.A., Assmann T.S., Duarte G.C., Gross J.L., Canani L.H., Crispim D. The role of the uncoupling protein 1 (UCP1) on the development of obesity and type 2 diabetes mellitus. Arg Bras Endocrinol Metabol. 2012 Jun; 56(4):215-25.
  2. Long J.Z., Svensson K.J., Bateman L.A., Lin H., Kamenecka T., Lokurkar I.A., Lou J., . . . Spiegelman B.M. 2016 Jul 14; 166(2):424-35. doi: 10.1016/j.cell.2016.05.071.

 

About

You may also like...

One thought on “Shoelaces and Uncoupled Respiration

  1. Dr. C

    January 31, 2017 at 11:24am

    Bahahahaha…Barry Allen.

    Permalink  ⋅ Reply
  2. Melanie Goetz

    February 1, 2017 at 2:56pm

    My first thought was why mice transfected with GFP was used as a control! I also found the calorimetry results very interesting. If mice with PM20D1 had a higher energy spending despite the same exact diet between the PM20D1 mice subjects and control GFP mice subjects, I wonder if this could have negative implications if too much fat was being invested into energy consumption rather than energy storage. In regards to clinical applications, would drug therapies involve the activation PM20D1 and do you think that this mechanism would need to be regulated?

    Permalink  ⋅ Reply
    • Author

      Barry Allen

      March 5, 2017 at 7:50am

      It’s a good point why use GFP as a control, but I feel that’s the best option to get confirmation the cells were successfully transfected without altering normal cell behavior. I definitely feel that the fat consumption was diverted more towards energy production apart from storage. It doesn’t specifically say it in the article but it seems that PM20D1 acts as an activator for energy production.
      For potential therapies PM20D1 would have to be activated but definitely need to be regulated. There was no indication if there was an amount of fat that could oversaturate PM20D1 so I imagine this therapy would be administered with perhaps some dietary constrictions as well. Plus with all this heat being produced, homeostasis would have to be monitored.

      Permalink  ⋅ Reply
  3. Brock

    February 4, 2017 at 8:53pm

    It was interesting to see how the authors were taking a new approach on trying to solve the quixotic problem of losing weight fast and easily. Though their work on PM20D1 and the N-acyl amino acids was interesting, they themselves admitted that there were issues when they tried to implement the system in vivo in mice. While the side effects they observed are much less alarming than the fat liquefaction that is observed with other uncoupling drugs like DNP, they’re still a considerable barrier to implementing a therapy in humans. Even if their therapy does eventually make it to market, will it really even help prevent obesity and obesogenic disease? Dissipating extra energy to the environment via uncoupling may keep people from making it to an episode of “My 600 lb. Life”, but uncoupling therapies cannot undo the damage done by bad dietary and exercise habits, which can quickly cause other diseases such as atherosclerosis and cardiovascular disease. While it’s definitely cool to think about uncoupling being used as a therapy, I believe that we already know the best way to treat obesity and obesogenic diseases and that’s through the less glamorous treatment of dieting and exercise.

    Permalink  ⋅ Reply
    • Author

      Barry Allen

      March 5, 2017 at 8:01am

      Brock you have an excellent point here. There are significant headway to be made before this kind of therapy could be made available of the market. Even if it does, I don’t foresee PM20D1 being the solution to obesity as whole. I feel this might help with those that may have a genetic predisposition to obesity, but in the end there as so many other factors (mainly eating habits and exercise) that have a much bigger influence. If PM20D1 therapy does become a major thing, I imagine doses can only be so high. and therefore be only able to do so much.

      Permalink  ⋅ Reply
  4. Brock

    February 5, 2017 at 6:21pm

    It was interesting to see how the authors were taking a new approach on trying to solve the quixotic problem of losing weight fast and easily. Though their work on PM20D1 and the N-acyl amino acids was interesting, they themselves admitted that there were issues when they tried to implement the system in vivo in mice. While the side effects they observed are much less alarming than the fat liquefaction that is observed with other uncoupling drugs like DNP, they’re still a considerable barrier to implementing a therapy in humans. Even if their therapy does eventually make it to market, will it really even help prevent obesity and obesogenic disease? Dissipating extra energy to the environment via uncoupling may keep people from making it to an episode of “My 600 lb. Life”, but uncoupling therapies cannot undo the damage done by bad dietary and exercise habits, which can quickly cause other diseases such as atherosclerosis and cardiovascular disease. While it’s definitely cool to think about uncoupling being used as a therapy, I believe that we already know the best way to treat obesity and obesogenic diseases and that’s through the less glamorous treatment of dieting and exercise..

    Permalink  ⋅ Reply
    • Author

      Barry Allen

      March 5, 2017 at 8:01am

      Brock you have an excellent point here. There are significant headway to be made before this kind of therapy could be made available of the market. Even if it does, I don’t foresee PM20D1 being the solution to obesity as whole. I feel this might help with those that may have a genetic predisposition to obesity, but in the end there as so many other factors (mainly eating habits and exercise) that have a much bigger influence. If PM20D1 therapy does become a major thing, I imagine doses can only be so high. and therefore be only able to do so much.

      Permalink  ⋅ Reply
  5. Gianna Barres

    February 26, 2017 at 9:17pm

    Hi Nick! Only knew it was you because of “why do science”. My question is similar to Brock’s in that the treatment here seems like another loophole for weight loss with questionable health improvement. In other words, this implies that people consuming high fat diets will stay skinny but be lacking the right nutrition. I was wondering, though, if you think that this might be useful for people that suffer from metabolic disorders in which avoiding weight gain is near impossible even while maintaining a healthy diet? So I guess these patients might be able to upgrade thermogenesis to promote weight loss so as to avoid obesogenic disease even though they might have already have relatively healthy diets? Maybe this is a less superficial implication for this kind of a therapy.

    Permalink  ⋅ Reply
    • Author

      Barry Allen

      March 5, 2017 at 8:09am

      I do agree with you and Brock that this proposed method of therapy does seem like an easy way out to losing weight but I envision this kind of therapy would be more reserved for those with a genetic predisposition (such as a metabolic disorder you mention) as opposed to just being a new fad to quickly lose weight. But even so I don’t feel the therapy is strong enough to just stand on its own.

      Permalink  ⋅ Reply
  6. Renan

    February 27, 2017 at 4:22pm

    I am one of the people who eats a lot and still skinny and I burn fat quickly just by sitting down and thinking about typical things!Is it my fast metabolic rate? Doctors told me I could be very bad for the economy if i happen to become a robot someday.
    Do you think the enzyme that might be causing this weight loss in mice is same enzyme that is causing me to have a fast metabolic rate? And if it is same enzyme or related to it, could that enzyme have been genetically built in my system or could it be the cause of the diet I have consumed in the past?

    Permalink  ⋅ Reply
    • Author

      Barry Allen

      March 5, 2017 at 8:19am

      I think you are definitely one of those people where others wish they had your metabolism. I cannot say if this particular enzyme is responsible for your metabolic rate. Is it possible? Sure, anything is possible in the field of science. Is it likely thought? Probably not, there are several different explanations that could be taken. I guess a starting question is if a lot of your diet is high in fats and if n-acyl amino acids are present in your blood.

      Permalink  ⋅ Reply
  7. Katlyn Mayer

    February 27, 2017 at 4:22pm

    I thought this was a very interesting article, Nick. As we learned in lecture, amino acids can undergo post-translational modifications by fatty acids, and this is a very cool application of that concept. I find it absolutely fascinating that even with a high fat diet, the mouse model of this enzyme allowed the mice to lose weight. The form of uncoupling in this paper seems safer than using a synthetic molecule like DNP, but at the same time, I could see this type of weight loss therapy being abused. What do you think are the ethical implications of this study?

    Permalink  ⋅ Reply
    • Author

      Barry Allen

      March 5, 2017 at 8:27am

      Seeing as we constantly would rather use a natural product over a synthetic one, PM20D1 does have that benefit. As for potentially being abused, I’m sure that is definitely a possibility. There is still work to be done to note all the effects it may pose on the body since it was mainly studied in a mouse living system. Depending on any side reactions would ultimately decide what kind of restrictions might need to be put in place to make sure no unnecessary harm falls to the victim. There are those cases where people abused diet pills and other supplements so I would not be surprised if that would be the case here.

      Permalink  ⋅ Reply
  8. Aisha Kourouma

    March 3, 2017 at 9:50pm

    Hi Nick! Fast weightloss is the craze these days so this was interesting to read. Hopefully this doesn’t give people false hope in thinking that this is an easy breezy way to lose weight. This also expressed some issues when trying to implement this treatment in vivo with mice. However, this route seems to be safer than synthetic options offered in the market today. It was interesting to see that amino acids can work together with fatty acids to go through post-translational modifications.

    Permalink  ⋅ Reply
    • Author

      Barry Allen

      March 5, 2017 at 8:34am

      It is true that companies are trying to make fast appealing ways to market weight loss, but with the issues of finding a way to effectively introduce this enzyme in a foreign path, I think we’re a little ways off from decided how effective it might be in a therapy sense. I do feel that if the field ever does make it as far as a marketable therapy, it only has so many capabilities and therefore would be heavily tethered to dieting and exercising. Everything can’t be that easy.

      Permalink  ⋅ Reply

Your email will not be published. Name and Email fields are required