Insulin may behave completely differently than what scientists thought. According to a new paper, the protein forms not three but six types of clusters, and this likely affects how quickly it works when given as a medication. The discovery could be used to finetune insulin shots for the millions of people living with diabetes.
Previous studies showed that insulin exists primarily as monomers, dimers, or hexamers. However, those studies looked at the average behavior of millions of proteins, which misses a lot of detail. Now, using single-molecule microscopy, Nikos Hatzakis and Knud Jørgen Jensen at the University of Copenhagen and their team have managed to get a much more refined picture, which they recently published in Communications Biology (1).
The team flushed a special microscope slide with a low concentration solution of insulin proteins, each tagged with a glowing fluorescent dye. Some of the proteins attached to the slide, while others continued to float in the solution and occasionally formed clusters with the attached proteins. By using a microscope technique that only lights up proteins right at the slide’s surface, the researchers followed along by watching how the amount of light changed when a tagged protein joined or left the cluster.
They recorded more than 2,300 clusters forming and breaking apart nearly 50,000 times. However, instead of only the three expected types of insulin clusters, they saw trimers, tetramers, and pentamers too. Due to this and an abundance of hexamers, the effective monomer concentration was significantly lower than expected. They also observed that insulin moves between the different cluster sizes mostly by adding monomers, not dimers as previously thought.
This is important because clustering affects the onset and duration of the protein’s effect as a medication (2). The monomer is the active form, and larger clusters must fall apart to work, meaning that they take longer to kick in after injection. In other words, the clustering directly influences whether a formulation is fast or slow-acting and when people with diabetes can take their shots.
“The current data would [ideally] be combined with experiments in cells and organisms. That is, of course, easier said than done.”
- Nikos Hatzakis, University of Copenhagen
The team believes that the newly observed clustering behavior is a direct result of the experiment requiring a much lower concentration of insulin than used in other studies, which also better approximates the concentration of insulin in the body. “We now have a far more detailed understanding,” said Jensen.
Unravelling these processes is a longstanding challenge, said Matthew Webber, a chemical and biomolecular engineer at the University of Notre Dame, who was not involved with the study. While the fluorescent labelling approach might not be suitable for all insulin formulations, “the knowledge revealed in this study will nevertheless inform the design of these new insulin variants.”
Hatzakis underlined that while the work is the most detailed to date, it has so far only been done outside the body. “The current data would [ideally] be combined with experiments in cells and organisms,” he said. “That is, of course, easier said than done.”
However, it isn’t impossible; advanced lattice light-sheet microscopes would allow the team to do just that, and Hatzakis plans to try one out in the near future. “I would then like to see how we can develop it into a general tool for evaluating new insulin drugs,” said Jensen.
- Bohr, F., Bohr, S.S.R., Mishra, N.K., González-Foutel, N.S., Pinholt, H.D., Wu, S., Nielsen, E.M., Zhang, M., Kjaergaard, M., Jensen, K.J. and Hatzakis, N.S. Enhanced hexamerization of insulin via assembly pathway rerouting revealed by single particle studies. Communications Biology, 6, 178 (2023)
- Hirsch, I.B., Juneja, R., Beals, J.M., Antalis, C.J. and Wright Jr, E.E. The evolution of insulin and how it informs therapy and treatment choices. Endocrine reviews, 41, 733-755 (2020)