The discovery came as a surprise, given that the chemical isolated by the biologists is not directly involved in regulating glucose production in the liver. Instead, it affects the activity of a key protein that controls the internal mechanism of our biological clock (circadian rhythm, i.e. daily night and day activities).
The idea that diabetes and obesity could be linked to the circadian rhythm is not a new one. It has been demonstrated that lab mice with altered biological clocks, often become obese and develop diabetes. A team led by Steve Kay, dean of the Division of Biological Sciences at UC San Diego, discovered the first biochemical link between the biological clock and diabetes two years ago, when he found that cryptochrome, a key protein that controls the biological clocks of mammals, insects and plants also controls glucose production in the liver and that the health of diabetic mice could be improved by changing the levels of this protein.
Kay and his team now discovered a small molecule that can easily be developed into a drug, which regulates the complex timekeeping mechanisms of cryptochrome in such a way that it is able to suppress glucose production in the liver. Humans and other mammals have evolved biochemical mechanisms that feed the brain with a steady supply of glucose when no food is consumed, during nighttime or during other times of inactivity.
Kay explains: "At the end of the night, our hormones signal that we're in a fasting state. And during the day, when we're active, our biological clock shuts down those fasting signals that tell our liver to make more glucose because that's when we're eating."
Diabetes is a metabolic disorder caused through an accumulation of glucose within the blood. The disease can lead to kidney failure, blindness, heart disease and stroke. There are two forms of diabetes, type 1 diabetes, in where blood sugar levels are elevated due to the destruction of insulin producing cells in the pancreas, and type 2 diabetes, which accounts for almost 90% of all diabetes cases, in which blood sugar levels are elevated because of a gradual resistance to insulin due to obesity or other problems.
In 2010, Kay and his team discovered that cryptochrome has a vital impact in controlling the body's internal timing processes, like the timing of the eating cycle patterns during the day and the timing of fasting during nighttime to maintain a steady supply of glucose in the bloodstream. Other recent studies have found that cryptochrome can also potentially reduce high blood sugar caused by asthma medication by adjusting the time at which the medication is taken. Kay commented: "We found that if we increased cryptochrome levels genetically in the liver we could inhibit the production of glucose by the liver."
In their latest study, Kay and his team discovered a much smaller molecule, named "KL001" after the first such compound identified by the Kay Lab, which can also control this activity. KL001 is able to slow down the biological clock by stabilizing the cryptochrome protein, meaning it basically prevents crypotochrome from being sent to proteasomes, the cellular waste bin. KL001s discovery occurred as a complete surprise during a parallel effort to identify molecules that lengthen the biological clock.
Tsuyoshi Hirota, a postdoctoral fellow in Kay's laboratory discovered a compound called 'longdaysin' 2 years ago, which had the so far greatest effect on the biological clock, by lengthening the daily circadian rhythm of human cells by over 10 hours. Hirota is currently continuing his research to find more chemicals that lengthen or slowed down the biological clock, in an effort to provide more insight into the intricate chemical and genetic workings of the circadian rhythm. Hirota and his Kay lab colleagues discovered KL001 by screening thousands of compounds from a chemical library with human cells in individual micro-titer wells that had luciferase gene from fireflies attached to the to the biological clock machinery, which gave off a glow whenever the biological clock was activated. Aside from discovering KL001, the biologists also discovered a number of other compounds during this process.
"We found other compounds that like longdaysin slowed down the biological clock. But unlike longdaysin, these compounds did not inhibit the protein kinases that longdaysin inhibits so we knew this compound must be working differently. What we needed to know was what is this compound interacting with? And we were absolutely stunned when we discovered that what was binding most specifically to our compound, KL001, was the clock protein cryptochrome that our lab has worked on in plants, flies and mammals for the last 20 years."
The team collaborated with biological chemists in Peter Schultz's laboratory at The Scripps Research Institute to characterize the compound and to gain a better chemical insight of its affect on cryptochrome to lengthen the biological clock.
Kay explained: "Those biochemical studies showed us that KL001 prevents cryptochrome from being degraded by the proteasome system, which was another big surprise. It essentially interferes with the signal to send cryptochrome to the garbage can."
The team also collaborated with Frank Doyle and his UC Santa Barbara team to gain a better understanding of the mechanism in which KL001 works with cryptochrome in regulating the biological clock. Kay commented: "They constructed a beautiful mathematical model of cryptochrome's role in the clock. That model was essential in allowing us to understand the action of the compound because the biological clock is very complicated. It's like opening the back of a Rolex and seeing the hundreds of tiny little cogs that are tightly integrated."
By using this mathematical model, the researchers were able to predict that adding KL001 to mouse liver cells should stabilize cryptochrome and that the higher cryptochrome level would block the production of liver enzymes, which stimulate the gluconeogenesis process, i.e. the generation of glucose during fasting. Their predictions were confirmed through experiments conducted in conjunction with David Brenner's lab. Brenner is dean of the UC San Diego School of Medicine and Vice Chancellor for Health Sciences.
"In mouse liver cells we showed that KL001 inhibited gene expression for gluconeogenesis that is induced when exposed to the hormone glucagon, which promotes glucose production by the liver. It's a hormone we all produce in fasting states. And our compound, in a dose dependent way, inhibits hepatic gluconeogenesis, the actual production of glucose by those liver cells."
Kay concludes saying their next step will be trying to understand the workings of KL001 and similar molecules, which affect cryptochrome function in living systems like laboratory mice. They also plan to investigate how compounds like these affect other processes, apart from the liver, which may link the biological clock to metabolic diseases. He adds: "As with any surprise discovery this opens the door to more opportunities for novel therapeutics than we can currently imagine."
Written by Petra Rattue