Using an interdisciplinary approach (molecular biology, biochemistry, neuroscience and systems physiology) research is focused on understanding how prolonged sleep deprivation of rats results in the development of many pathologies including hyperphagia, loss of body weight, and increased energy metabolism.
Rat Model of Chronic Sleep Deprivation/Restriction to Investigate Metabolic Dysfunction
When humans or animal models such as rats are subjected to chronic sleep deprivation or restriction (SD/SR), many pathologies develop. Most of them can be characterized as dysfunctions of normal metabolic processes. For example, chronic SD/SR leads to hyperphagia (increased food intake behavior) but loss of body weight, significant changes in hormone profiles, changes in autonomic nerve activity, alterations in reproduction function, and many others. My research focuses on many of these pathologies.
Despite doubling of food intake, body weight progressively declines.
A. Biochemical basis of SD/SR hypermetabolism
Chronic SD/SR results in a substantial increase in metabolism. Biologically, this poses an interesting problem in that SD/SR hypermetabolism does not make sense because it is not adaptive. Just because the subject is sleep-deprived, why should that lead to a state of hypermetabolism?
We demonstrated previously that chronic SD robustly up-regulates uncoupling protein 1 (UCP1) in brown adipose tissue (BAT) mitochondria. This protein "uncouples" the thermodynamic energy of cellular respiration from making ATP, and instead, releases the energy as heat.
Metabolic rate, as measured by O2 consumption, increases by more than 50% (above). Much of the elevated metabolism can be explained by a substantial up-regulation of UCP1 mRNA (using RT-PCR) in BAT mitochondria (panels A and B).
Our current goals are to determine the cellular "upstream" events that lead to up-regulation of UCP1. Our working hypothesis is that "bottlenecks" of metabolism (i.e., non-equilibrium regulatory enzymes) are also up-regulated to increase carbon flow through metabolic pathways to fuel increased metabolism. An example is adenosine monophosphate protein kinase (AMPK), which been call the "cell's fuel sensor". We are also investigating enzymes that are at branch points of metabolic carbon flow within the cell. For example, one hypothesis is that chronic SD/SR leads to increased gluconeogenesis. The rationale for this hypothesis is that within days of SD, liver and skeletal muscle glycogen stores are rapidly mobilized and remain at very low levels. Moreover, body fat is also rapidly mobilized so that at necropsy, there is practically no fat tissue within the abdominal cavity. Thus, in order to continue to fuel cellular respiration, it makes sense that gluconeogenesis increases.
For techniques, we employ functional assays of enzymatic activity using spectrophotometric methods, and gene expression with RT-PCR and western blotting.
B. Neurobiological basis of SD/SR hyperphagia and hypermetabolism
This project is a collaboration with Dr. Gloria E. Hoffman. We investigated changes in neuropeptide neurotransmitters within the arcuate nucleus of the hypothalamus that help explain the mechanisms that govern food intake behavior during SD/SR. This work continues. We are also interested in how peripheral hormones that regulate food intake behavior, including insulin, leptin, ghrelin, and CCK play a role in SD/SR hyperphagia. Recently, we completed a study involving histidine decarboxylase, an enzyme of the posterior hypothalamus that regulates histamine synthesis. We are interested in how the enzyme and histamine maintain wakefulness, and how histaminergic neurons interact with other neuronal systems, but specifically sympathetic neurons that innervate BAT to regulate metabolism.
Techniques employed are hormone assays (e.g., ELISA) and immunocytochemistry, where specific analyte is localized using antibody, and the signal from the antigen-antibody complex is
enzyme-amplified and analyzed by microscopy.
These are sections of the posterior hypothalamus of rat brain, specifically the tuberomammillary nucleus (TMN). TMN neurons are stained for the enzyme, histidine decarboxylase (HDC), which is responsible for histamine biosynthesis. The release of histamine is one of the brain's mechanisms for keeping the subject awake. You know this anecdotally because if you take an anti-histamine drug for allergies, it will make you drowsy.
Rats were sleep-deprived for 5, 10, and 15 days; controls were home-cage rats. The dorsal aspect of the TMN is shown on the left panels; the panels on the right are of the ventral TMN. By day 5 of SD, HDC is substantially up-regulated compared to controls in both dorsal and ventral TMN, and remains elevated. This contributes to the rat being able to stay awake.
C. Physiological basis of onset of type 2 diabetes during chronic SD/SR
Our preliminary studies suggest that within just 2 weeks of sleep restriction (20 hours per day forced wakefulness, 4 hours per day sleep or rest in home cage), rats clearly exhibit signs of the onset of type 2 diabetes. This was determined by administering a glucose tolerance test (GTT), which is a standard clinical test performed on humans who are suspected of being diabetic. Although this is an important finding, we do not know if the signs of type 2 diabetes will be reversed upon obtaining period of normal sleep. Investigations continue.
Sleep-restricted and control rats were injected intraperitoneally (ip) with a bolus of glucose solution. Baseline and post-IP glucose blood samples were obtained and glucose levels were measured. 15 min after ip glucose injection, sleep-restricted rats had a significantly higher blood glucose compared to controls. This suggests that sleep restriction resulted in either decreased insulin and/or decreased ability of cells to transport glucose into cytoplasm.
D. What happens when sleep restriction is very chronic?
Let's face it, we live in a 24-hour world of instant communications, round-the-clock television and access to movies, electronic games, etc. Add to that everyday stressors such as problems at work and at home, money, health, etc. As a consequence, it is not surprising that most of us are chronically sleep-deprived, and trying to "catch up" on weekends may not be enough.
We asked a simple question: What happens to food intake and body weight when rats are chronically sleep-restricted? A cohort of rats was sleep-restricted for 20 hours per day, 7 days were week, for 60 days. They had 4 hours per day to sleep or engage in other behaviors in the home cages. Then, on day 61, we changed our paradigm to allow rats "weekends off". That is, they experienced 20/4 sleep restriction Monday through Friday but they were returned to their home cages on Saturday and Sunday; this weekly cycle were repeated until day 205!
Across 205 days of continuous or intermittent sleep restriction, rats ate 9.8% more chow compared to controls (P < 0.001) and consistently had 10% lower body weights (P < 0.001). There were occasional spikes or drops in food intake that could not be correlated to any changes in ambient temperature or other factors. The important conclusion from this study is that the loss in body weight never recovered, even with "weekends off."
PAPERS PUBLISHED IN PEER-REVIEWED JOURNALS
• Koban, M., L.V. Sita, W.W. Le, and G.E. Hoffman (2008). Sleep deprivation of rats: the hyperphagic response is real. Sleep 31:927-933.
• Koban, M., W.W. Le, and G.E. Hoffman (2006). Changes in hypothalamic CRH, NPY, and POMC gene expression during chronic sleep deprivation of rats. Endocrinology 147:421-431.
• Koban, M. and C.V. Stewart (2006). Effects of age on recovery of body weight following REM sleep deprivation of rats. Physiology and Behavior 87:1-6.
• Koban, M. and K.L. Swinson (2005). Chronic REM-sleep deprivation of rats elevates metabolic rate and increases UCP1 gene expression in brown adipose tissue. American Journal of Physiology - Endocrinology and Metabolism 289:E68-E74.
• Swinson, K.L. and M. Koban (2005). FORMAzol® as an RNA storage medium: a cautionary note when performing RT-PCR. Journal of Biochemical and Biophysical Methods 63:149-153.
• Koban, M., A.A. Yup, L.B. Agellon, and D.A. Powers (1991). Molecular adaptation to the thermal environment. Heat shock response of the eurythermal teleost Fundulus heteroclitus. Molecular Marine Biology and Biotechnology 1:1-17.
• Kent, J.D., M. Koban, and C.L. Prosser (1988). Cold acclimation induced protein hypertrophy in channel catfish and green sunfish. Journal of Comparative Physiology B 158:185-198.
• Koban, M., G. Graham, and C.L. Prosser (1987). Induction of heat-shock protein synthesis in teleost hepatocytes. Effects of acclimation temperature. Physiological Zoology 60:290-296.
• Koban, M. (1986). Can cultured teleost hepatocytes show temperature acclimation? American Journal of Physiology 250:R211-R220.
• Koban, M. and D.D. Feist (1982). The effect of cold on norepinephrine turnover in tissues of seasonally acclimatized redpolls (Carduelis flammea). Journal of Comparative Physiology. B. 146:133-144.
SELECTED MEDIA COVERAGE
Please click on the links that highlight our research
• Research Spotlight in Morgan Magazine, Volume 1, 2012. Available at: Pursuing Mysteries of Reproduction
• "Poor sleep disturbs neuroendocrine control of appetite." Published online April 22, 2009 by New-Medical.net. Available at: http://www.news-medical.net/news/2009/04/22/48641.aspx
• "Evidence mounts that short or poor sleep can lead to increased eating and risk of diabetes." Published by online April 24, 2009 by ScienceDaily.com. Available at: http://www.sciencedaily.com/releases/2009/04/090421181032.htm
• "Staying awake: changes in brain histidine decarboxylase expression underlie wakefulness in chronic REM sleep deprivation." Expert commentary video interview with SleepClinician.com, 2009. Available at: http://www.sleepclinician.com/video/id/51/
• "Not enough sleep causes more eating and diabetes risk." Published online May 1, 2009 by Diabetes in control.com. Available at: http://www.diabetesincontrol.com/articles/diabetes-news/6675-not-enough-sleep-causes-more-eating-and-diabetes-risk