Summer Research 2014: GLUT1 Membrane Density and Activation
Evans Lodge (Larry Louters)
Carbon is cellular currency – without the sugars that sustain and power
every living organism’s metabolism, the earth would be void of life itself. This
summer, the Louters Lab continued its research of GLUT1, one of twelve passive
glucose transporters that can be found in almost all mammalian tissues. Although
the project this summer involved seven different students investigating GLUT1 from
a variety of angles, my personal research delved into the relationship between
concentration (the amount of GLUT1 on the surface of a cell at any given moment)
and activity (the amount of glucose transported into a cell by GLUT1). Although it
would make intuitive sense to assume a linear relationship between the two, past
research in the Louters Lab and literature evidence contradict such a claim.
My work in the lab involved a variety of different biochemical research
techniques, but I ended up relying almost exclusively on two for the majority of my
data. The first, a glucose uptake test, uses a radioactive sugar almost identical to
glucose that is transported into the cell in an identical fashion. By examining the
amount of radioactivity inside a cell after feeding it the radioactive sugar analog, I
was able to probe into the “activity” of GLUT1 on a cell at a given time. The second
technique, called flow cytometry, allows a cell-by-cell examination of a tissue
culture. By labeling the GLUT1 protein in a cell population before running them
through the cytometer, it is possible to find the exact amount of protein on each
individual cell and search for differences in the population as a whole (as well as
between different populations). By using these techniques, I was able to delve
deeper into the relationship between GLUT1 concentration and activity in two
different cell lines. One, L929 fibroblasts, are connective tissue cells harvested from
mice. With a low amount of GLUT1 present in each cell, they are extremely “activateable” and respond to a variety of pharmaceutical and physical stresses by increasing
the activity of GLUT1 on the cell surface. The other, HK2 human kidney cells, have a
large amount of GLUT1 on the cell surface and are much less responsive to any sort
of activators, pharmaceutical or otherwise.
Although my work will carry on into the coming school year, my current
results show a continued non-linear relationship between GLUT1 concentration and
activity. We’ve also noticed that cell activity is incredibly dependent on confluency
(the amount of cells in a tissue culture plate at any given moment). Although cells
that are closely packed into a plate (highly confluent) more closely resemble the
actual cell-cell interactions that occur in our bodies, we are interested in how GLUT1
activity increases and decreases depending on the proximity of individual cells. At
present, it appears that cells spaced widely apart (under-confluent) are significantly
more active than those pressed closely together. This, paired with our ability to
activate cells with different pharmaceutical agents and deactivate cells by inhibiting
necessary GLUT1 coding DNA, has allowed us to debunk several hypotheses from
the beginning of the summer. It appears that HK2 cells, thought to be much less
activate-able than L929s, are significantly more inducible at lower confluency.
We’ve also determined that the concentration of GLUT1 on the surface of a cell isn’t
the determining factor in the activate-ability of the cell (at least in HK2 cells). In
many ways, my work has broken down some assumptions from the past summer
and given us an even foggier vision of how GLUT1 really works. Our hope is that we
now know how to take a more holistic approach to studying GLUT1 in the future,
taking into account such disparate influences as cell density, glucose availability,
pharmaceutical activation, etc.
This summer has been incredibly helpful for me, a student interested in
chemistry, medicine, geography, public health, and everything in between. Aside
from the sheer breadth of new material I’ve learned this summer, I’ve loved the
chance to feel like a much greater part of the biochemistry department and
community. I’m still not sure where I’ll end up after graduation, but that isn’t
necessarily the point. I have a group of professors and fellow researchers that
support and encourage me along, and that’s been much more important to me than
anything else. I am incredibly thankful to Professor Louters for hiring me during the
summer, and I can’t wait to keep working with him during the coming year (or
years).