Discovery route: Path to potential diabetes drugs began with a simple question

More than 12 years of research led Anath Shalev, M.D. (right, with Junqin Chen, Ph.D.) from a basic discovery to the first human trial of a new type of diabetes drug.

In 2002, diabetes researcher Anath Shalev, M.D., asked a basic question: What gene in the insulin-producing islets of the human pancreas is most turned on by high levels of glucose, a hallmark of diabetes?

The answer has led the UAB endocrinologist to discover new cellular pathways in beta cells of the islets, pathways that are a key to diabetes progression or protection. Those discoveries have now opened the door to the first human trial of a potential diabetes drug with a mode of action different from any current diabetes treatment. (Learn more about the trial, which will begin in early 2015, in this story.)

Anath Shalev explains verapamil's protective effects against diabetes, and a new human clinical trial of the drug at UAB, in this video.

Beta cells are critical in type 1 and type 2 diabetes. In both diseases, the cells are lost gradually due to programmed cell death (apoptosis); but the trigger for that programmed death was unknown. The loss of beta cells contributes to the progression of diabetes, a growing worldwide epidemic that affects more than 20 million people in the United States, making it the seventh leading cause of death and the source of complications like blindness and more than 40,000 lower limb amputations a year.

From Molecular Mechanisms to New Treatments

The beta-cell gene that responded to the high glucose in Shalev’s 2002 experiment produces TXNIP (pronounced "ticks-nip"), a protein normally involved in controlling oxygen radicals in many types of cells but never known to be important in beta-cell biology. Its response to glucose was intriguing because TXNIP (thioredoxin-interacting protein) was already recognized as a regulator of thioredoxin. Overexpression of thioredoxin had previously been shown to prevent experimentally induced diabetes by inhibiting the programmed death of islet beta cells. Since TXNIP inhibits thioredoxin, and because Shalev had discovered that islet TXNIP was highly regulated by glucose, Shalev realized that TXNIP might have major implications for beta-cell biology.

What does it take to go from a basic microarray gene discovery to a human trial of a completely novel drug to treat diabetes?

A dozen years of elegant research unraveling the control and function of a protein called TXNIP.

Over the next dozen years, Shalev — who left the University of Wisconsin–Madison to head the UAB Comprehensive Diabetes Center in 2010 — set out to reveal how TXNIP acts in cells at the molecular level, knowing that an understanding of those molecular mechanisms might point to possible new diabetes treatments. The payoff has been substantial: Using cell cultures, mouse models and pancreatic islets isolated from humans, the Shalev lab team has shown that manipulating TXNIP levels up or down in beta cells could exacerbate or protect against experimental diabetes.

Details about the research journey show the incremental steps that basic science takes, and how those connected steps sometimes lead to potential clinical impacts.

Controlling TXNIP to Treat Diabetes

In 2005, the Shalev lab team found that beta-cell TXNIP levels are higher in mouse diabetes models, and that experimentally increasing TXNIP levels in rat beta cells in vitro led to increased programmed cell death, by means of a well-known trigger signal of apoptosis. The Shalev team also found that sugars in general, whether metabolized or not, turn the TXNIP gene on. This clue led them to a newly identified carbohydrate response element (ChoRE) in the TXNIP promoter that acts as a regulator of TXNIP.

In 2008, the Shalev lab developed mice that had little or no TXNIP in their beta cells. These lower levels protected against experimental diabetes. The team also discovered that the lower levels sent a known signal that inhibited mitochondrial beta-cell death. Shalev wrote, “These results suggest that lowering beta-cell TXNIP production could serve as a novel strategy for the treatment of type 1 and type 2 diabetes by promoting endogenous beta-cell survival.”

An Approved Drug Offers Protection

In 2012, the Shalev group tested an already approved oral drug that they had earlier found to reduce levels of TXNIP in heart cells. The drug — verapamil — is a calcium channel blocker used primarily to treat high blood pressure, but also to treat migraine headaches. Shalev’s team found that exposing in vitro beta cells or isolated human islets to verapamil reduced TXNIP expression, and halted programmed apoptotic death of beta cells. Furthermore, mice that were fed verapamil in their drinking water were protected from experimentally induced diabetes, and verapamil rescued mice that already had diabetes. The verapamil mice had lower TXNIP levels and less programmed beta-cell death, as well as better levels of insulin

"I actually went down to the mouse house to see if the mice were getting diabetes," Shalev told The Birmingham News in 2012. When she found normal glucose levels, "We were dancing."

In those studies, the group also revealed how verapamil lowers TXNIP — the decreased intracellular level of calcium ions caused by verapamil led to phosphorylation of the ChoRE binding protein that normally responds to glucose to control TXNIP transcription at the ChoRE. This phosphorylation prevented the binding protein from entering the beta-cell nucleus and interacting with the TXNIP gene. Shalev noted that these verapamil results identified, for the first time, “… an effective pharmacological means … to inhibit pancreatic beta-cell expression of proapoptotic TXNIP, enhance beta-cell survival and function, and thereby prevent and even improve overt diabetes and shed light on the mechanisms involved.”

Another Role for TXNIP, Another Drug Target?

In 2013, TXNIP was shown to play another crucial role in beta-cell biology when the Shalev laboratory team discovered that high levels of TXNIP directly blocked insulin production in beta cells, acting through a newly identified pathway. TXNIP, they found, induced a microRNA called miR-204, which in turn down-regulated the MAFA transcription factor involved in promoting transcription of the insulin gene.

This means that miR-204 may offer another target for a future RNA drug, an area that is currently also being actively pursued by the Shalev lab. MicroRNAs, with 20 to 24 noncoding nucleotides, have rapidly gained prominence as regulators of gene expression in health and disease. Researchers are beginning to explore whether silencing targeted microRNAs may lead to a treatment for cancers or other diseases.

TXNIP's Vicious Cycle

This year Shalev reported that TXNIP — surprisingly — can induce its own transcription. Her UAB research team found that TXNIP does this by affecting the same ChoRE binding protein (ChREBP) that was previously found to be key in the response to the drug verapamil. The researchers experimentally elevated TXNIP levels in beta cells and found this caused decreased phosphorylation of ChREBP, which led to its increased entry into the nucleus and its increased binding to the TXNIP promoter to boost transcription. This creates a harmful positive-feedback loop.

"These findings support the notion,” Shalev wrote in this 2014 paper, “that TXNIP levels rise over time, not only as a result of elevated blood glucose levels and/or endoplasmic reticulum stress, but also as part of a vicious cycle by which increased TXNIP levels lead to more TXNIP expression and thereby amplify the associated detrimental effects on beta-cell biology including oxidative stress, inflammation, and ultimately beta-cell death and disease progression.”

First Human Trial

Get a quick overview of the science behind UAB's verapamil
trial in this animation

The story doesn’t end here. Shalev’s long trail of laboratory research has now led to the first human trial to see if verapamil has an effect in patients who have developed type 1 diabetes within the previous three months. Adult volunteers, ages 19-45, will be treated with verapamil or a placebo for one year, as their insulin and blood glucose levels are continuously monitored. The three-year, $2.2 million trial will be conducted by the UAB Comprehensive Diabetes Center with funding from JDRF, the largest charitable supporter of type 1 diabetes research.

Meanwhile, a UAB partnership with the Southern Research Institute — called the Alabama Drug Discovery Alliance — is already working to develop small therapeutic molecules that mimic the diabetes-protecting effect produced by verapamil and inhibit TXNIP, but have a greater selectivity and efficacy. [Learn more about this work, and other high-potential projects in the Alabama Drug Discovery Alliance, in a new feature from UAB Magazine.]

So Shalev’s simple question — what gene in insulin-producing beta cells is most turned on by glucose? — has thus led the research out of her laboratory to possible new drugs, acting against a novel target to alleviate or reverse diabetes.

— Jeff Hansen

Alcohol throws off circadian clock to damage liver

Having evolved to keep time with our planet's rhythms – day and night, light and dark – we are wired at the genetic level to sleep at night and to wake and eat during the day. Research in recent years revealed that genetic and protein feedback loops – or clocks – operate in 24-hour cycles in every human cell. The clocks signal to thousands of genes, many of which speed up our ability to make and use energy from food during the day and turn it down at night.

Bucking those patterns – say by working the night shift – has been shown to increase a person’s risk for heart disease, diabetes, cancer, depression, etc.

In a new twist, Shannon Bailey, Ph.D., associate professor in the Division of Molecular and Cellular Pathology within the UAB School of Medicine, just published a study that found chronic alcohol use may interfere with the genetic clocks in liver cells to accelerate liver damage. Dr. Bailey is a longtime liver disease expert with a new research focus on the role of circadian clocks in alcohol-related liver damage.

We thought to ask her whether too many martinis can throw off molecular clocks and, from a circadian point of view, what the healthiest hour is to drink a glass of wine.



Show notes for the podcast:

1:51 Genes are long chains of molecules that encode instructions for the building of the proteins, the workhorse molecules that make up bodily structures and signals. Interestingly, the process of turning genes into proteins proceeds at a certain rate, so it has become the basis of a system that keeps time like a clock. To achieve a biochemical balance necessary for life, many genes are part of pathways that sense when there enough of any given protein, and sends signals to shut down the building process: so-called feedback loops. The twenty or so genes and proteins that make up the human circadian clock happen to perform these loops in a roughly 24-hour cycle, and so evolution favored them. Creatures that happened to align their metabolism to these clocks became one with their environment and were more likely to survive. Thus, the cells making up most life on earth today -- bacteria, plants, animals, etc. -- include genetic clocks.

3:44 In a larger sense, the clocks show the ability of genes to adjust their action in the face of changing environment. Genetic changes in energy use also occur after meals whenever they occur, and during the flight or fight response.

4:35 One of the functions of the circadian clocks in every human cell type is to turn off metabolic pathways that produce cellular energy from food when we don't need them. While obviously vital to life, highly active metabolic pathways create byproducts like free radicals that tear apart sensitive cell components and cause cells to self-destruct as part of many diseases, including major ones related to energetics: diabetes and heart disease. Shutting them down at night may help us to live longer.  

5:33  A major focus of Dr. Bailey's research is the mitochondria, sub-compartments of human cells that convert sugar from food into cellular energy by using oxygen.  She is especially interested in the role of mitochondria in liver disease. Overproduction of free radicals by mitochondria in liver cells damages other parts of the same cells, and shutting down these pathways at night may give cells a chance to repair the damage. A key emerging question is how not circadian clock genes in the nuclei of liver cells signal to mitochondria to control energy production.

6:39  While most studies look at the effect of staying up at night on circadian biology, Dr. Bailey wanted to look at the effect of alcohol on circadian clocks. Some of her interest stems from the fact that neuroscientists have been exploring in recent years whether or not circadian clocks in nerve cells in the brain may contribute to the forming of addictions. Up until the current study, only a few studies had looked at the effect of alcohol consumption on peripheral cells (gut, heart, liver, etc.). It's really starting to take off, says Dr. Gohlke.

8:43 As whole, the body’s circadian clock is regulated by a part of the brain called the suprachiasmatic nucleus, which drives daily physiological and behavioral rhythms. The newest frontier, embodied by Dr. Bailey’s study, is the effort to understand the role of the circadian clock in each cell type, and what happens when the cell-specific clocks are out of sync with the central clock in the brain. The liver is the organ in the body most responsible for regulating system-wide energy needs and is charged with holding steady levels of sugar supplied to cells by the bloodstream. Studies have shown that molecular clocks in the liver help keep the blood sugar level constant as we eat and fast, sleep and wake., largely through adjusting levels of the hormone insulin. If we throw the clocks off, we throw our blood sugar off and contribute to the development of diabetes.

9:54  The liver also plays a prominent role in the amount of cholesterol and fat in the blood, as well as the breakdown of drugs in the bloodstream.  Should the clocks be shown to regulate those pathways and alcohol affects them, then chronic drinking hardens arteries, the leading cause of heart attacks and strokes, and contributes to obesity.

10:32 To study the effect of alcohol on circadian clocks, Dr. Bailey and her team separated mice into two groups, one that received a healthy diet, and a second that had the same diet plus a steady supply of alcohol (ethanol). They then collected brain and liver samples for both mice to look for changes in clock genes patterns. They found that expression of metabolic genes that normally up and down  in 24 hours cycles no longer do so with chronic alcohol use. It also looked like metabolic pathways that normally operate in sync became disjointed.

13:35 In a healthy individual, certain clock genes in liver cells are expressed three hours after the same genes are expressed in the brain. One theory holds that the time lapse is a temporal signal between the systems that monitor how much energy we need, and those that supply the right amount of energy in response.  If alcohol throws off the liver cell clocks, they may no longer proceed in sync with the brain clock. Such a loss of synchrony represents a potential disease mechanism in heart disease, diabetes and obesity.

15:56 Fatty liver disease, which is very common in the United States, is the earliest stage in the progression toward much more serious liver diseases like cirrhosis. It is also seen people in pre-diabetes or obesity. Healthy people store their fat in their fat cells, their adipose tissue, and not their liver cells. Dr. Bailey is interested in whether or not disruption of liver clocks contributes to this build up of fat in liver cells.

17:31 As the understanding of molecular clocks grows, researchers will seek to influences parts of the clock that contribute to disease. Research teams have already shown that some drugs, at least in animal models, can fine-tune clocks to counter weight gain. Dr. Baily is looking forward to testing whether such drugs can counter the contribution of clock genes, under the action of alcohol, to liver damage

18:03 Dr. Bailey's study did look at whether the expression of proteins that break down alcohol fluctuate with time of day, but not at how active those proteins are. She has proposed a series of studies that will seek to determine what time of day is best for that glass of wine. The studies so far at least suggest that there may be times of day when the liver is more or less vulnerable to the toxic effects of alcohol.