An in vitro model of insulin resistance and its reversal
Mesenchymal stem cells capable of differentiating into mature adipocytes (fat cells, Fig. 1) can be obtained from small fat biopsies collected at different times of the year. These can then be cryopreserved to provide us with a ready supply of material for study and manipulation.
We've discovered that bear cells obtained in hibernation are insulin resistant, just like the bears themselves. Interestingly, this can be reversed by culturing the cells with serum from the active season. The active serum constituent remains to be identified but appears to be a protein.
Former students Jamie Gehring and Kimberly Rigano along with Brandon Hutzenbiler worked tirelessly to develop this model.
Currently, in collaboration with Dr. Joanna Kelley (WSU) and Michael Saxton,we aim to identify key regulators of the insulin sensitivity switch.
Fat cells secrete numerous factors including leptin and adiponectin which have major effects on energy metabolism, food intake, and other functions. Their roles in modifying adipose function remains to be clarified.
Figure 1. Cultured bear adipocytes stained with Oil Red-O to identify lipid droplets.
A word cloud representing the various aspects of research being performed in the Jansen lab.
Role of circadian rhythms in bears
Rodents and other small hibernators lose their circadian rhythms when undergoing torpor bouts. However, bears continue to express behavioral and cellular circadian rhythms during hibernation. While it might seem energetically wasteful to express these rhythms, the fact that they continue likely means they serve an important purpose. One such role may be in separating cellular process that are incompatible with one another (e.g., oxidation-reduction). Another may be to fine tune glucose or fat utilization (the body's main fuels). A third may be to minimize oxygen consumption.
The reduced amplitude of circadian rhythms in hibernating bears likely reflects the ability to modulate many different process without having to dispense with rhythms altogether. Figure 2 shows the difference in rhythm features of glucose uptake by culture adipocytes from hibernating bears (blue) and bears that were fed during hibernation (red). The increased glucose uptake by cells after feeding likely reflects a metabolic switch in response to newly available nutrients.
This work was performed by Kaylie Shaver, a fourth year veterinary student and recent veterinary scholar at WSU.
Figure 2. Rhythms of glucose uptake by cultured bear adipocytes during hibernation or when fed glucose (red) during hibernation. Note the increase in rhythm amplitude and total glucose uptake (inset) following feeding.
Polar bears are the closest relatives of brown bears yet they do not hibernate unless pregnant. However, similar to brown bears, we recently discovered that polar bears continue to express circadian rhythms even while fasting during the late summer-early fall (Fig. 3). We hypothesize that maintaining these rhythms allows bears to optimize energy expenditure.
Changes in amount of sea ice from which polar bears can hunt is of major concern to biologists as it is unclear if they can adapt to terrestrial foods.
To see how much ice is being lost annually, and in increasing amounts over the past 40+ years of measurements, please visit the National Snow and Ice Data Center News and Analysis webpage.
This work is being performed in collaboration with Drs. Jasmine Ware (Gov't of Nunavut, Canada), Karyn Rode (USGS) and Tanya Leise (Amherst College).
Figure 3. Annual patterns of activity of 4 free-ranging polar bears. These actograms are plotted as follows: each line represents two consecutive days, with the second day plotted underneath the first, and so on. The black ticks on each line represent an activity bout with the height of the tick proportional to the amount of activity.
Cellular energetics and metabolic fuel switching
Cells and tissues depend on the nutrients that are ingested or synthesized by the body to survive. Fat is used to store energy. During hibernation or prolonged starvation, resources are limited and alternative fuels such as fat are used. Bears accumulate tremendous amounts of fat before hibernation. Then, during hibernation bears are mostly inactive and do not eat for up to 5 months. Yet, bears don't lose muscle mass (known as atrophy) or bone density and their kidneys are operating at minimal levels. Similar periods of starvation in humans lead to death. For bears to survive this prolonged fast must require major adjustments in fuel utilization, oxygen consumption, energy expenditure or all three.
Bears have evolved the capacity to survive these extended periods of fasting but the exact mechanisms have not been fully revealed. Our laboratory is using a cell culture system or "hibernation in a dish" to study this. Much like insulin resistance, hibernation cells also display many similarities in metabolism to the bears themselves.
Figure 4 shows the results of a phenotype test performed with the Seahorse XFp flux analyzer and confirms that a cell culture system can be used to measure and manipulate cellular energetics.
This work was the focus of Hannah Hapner's Master's project with the assistance of Brandon Hutzenbiler. A veterinary student, Grace Sun, also worked on fuel sources and energetics by examining the role of fatty acid saturation on lipolysis (Fig. 5) and ATP production in adipocytes (Fig. 6).
Figure 4. Rates of glycolysis (x-axis) and mitochondrial respiration (y-axis) in cells from hibernating bears (blue), active season bears (red) and bears fed during hibernation (gray). Note the lower rates of glycolysis and oxygen consumption in hibernating cells compared to active cells.
Figure 5. Measurement of lipolysis product, glycerol, in hibernation bear adipocytes cultured in bovine serum albumin (BSA) only (CONT) or in physiologically-relevant concentrations of palmitic acid (unsaturated), oleic acid (monounsaturated) or EPA (polyunsaturated) followed by stimulation with isoproterenol (ISO) to mimic norepinephrine. Note the general decrease in glycerol production with increasing degrees of unsaturation.
Figure 6. Rates of ATP production in hibernation adipocytes before and after ISO stimulation. As lipolysis is an energy-dependent process (see Fig. 5), stimulation results in a decrease in ATP production. However, the polyunsaturated fatty acid, EPA, blunts this effect