The missing links in insulin action

When David James did his PhD at the Garvan Institute in Sydney sometime back in the last millennium, he was one of only two postgraduate students and the institute was part of St Vincent's hospital laboratories.

In 2001, and after establishing himself as an internationally respected leader in diabetes and cell biology research, James returned to the Garvan to head its diabetes and obesity research program, in a newly fitted-out and vibrant research community comprising around 400 scientists and over 70 PhD students.

There are now about 70 people at the Garvan working on type 2 diabetes, which happens to be one James' long term interests. According to James, one important element about the program he heads is that it comprises independent groups each working on a different aspect of diabetes research. In addition, the program spans the whole breadth of focus, from basic science right through to clinical research using the latest technologies.

The program encompasses three different methodologies. Firstly, the researchers are trying to understand how insulin works under normal circumstances, because, as James puts it, "to figure out how the thing gets broken in disease, you have to know how it works normally". About two years ago, James came to the realisation that clinical science did not know as much about insulin action as was thought.

"I think a lot of people figured that insulin action was just about solved - insulin controls glycogen synthesis, glucose uptake, lipid synthesis and so on, and we just needed to understand those processes," he says.

James' group therefore decided to take a different and broader look at the issue. They focused on protein phosphorylation because most of the actions of insulin seem to involve this mechanism at some level.

Using stable isotope labelling and quantitative mass spectrometry, they screened different samples and cell populations to find novel phosphorylation events that occurred in response to insulin.

"We sequenced the phosphopeptides that came out of the screen (quite a list) and much to our surprise, found three to four that really captured our attention. These proteins provided a glimpse into what is clearly a much broader array of insulin actions than we thought." This work is soon to be submitted for publication.

At the Hunter Cellular Biology meeting, being held in the Hunter Valley this week, James will talk about two of these proteins that undergo substantially increased phosphorylation in response to insulin, neither of which were previously linked to growth factors.

Both proteins seem to play an important role in RNA stability, and particularly in microRNA (miRNA) processing. Such a role offers a whole new level of regulation of the insulin response, at the level of gene expression and translation. Growth factors such as insulin could regulate either the levels of message (mRNA) or the accessibility of message to the translational machinery in a very acute way.

"It is also possible that insulin signalling itself regulates miRNA processing, which is really interesting and exciting for us," James says. "We now want to figure out the nuts and bolts of this - what messages are being turned on and off by this pathway? That is going to probably take us into miRNA metabolism and other such areas that are a bit scary for a cell biologist."

More exciting results on one of these insulin-responsive phophoproteins have also come from a knockout mouse tracked down from a German thesis by one of James's PhD students. Testing by his group revealed these animals to have profoundly decreased muscle and adipose tissue mass compared to controls.

"This is very exciting because it suggests that insulin might regulate the differentiation or growth of these cells in a manner that is somehow dependent on miRNA processing or something like that- we don't really know yet. The results with this mouse might also take us down other avenues vis-a-vis the disease."

---PB--- Insulin resistance and disease

The second research focus of the diabetes and obesity program is the connection between insulin resistance and disease. This phenomenon called insulin resistance is clearly one of the earliest abnormalities to precede disease with respect to type 2 diabetes.

Insulin resistance is defined as the inability of insulin in the body to work correctly in peripheral tissues. There is an enormous amount of interest in insulin resistance because the feeling, pharmacologically, is that drugs that prevent it from happening might substantially delay the onset of disease.

"At the moment, there are many efforts and drugs out there that target the complications of the disease once it has taken hold, but that is not where we want to be," James says. "It is like putting bandaids on a wound that is never going to go away."

Recent work in the program has provided novel insights into the mechanisms of insulin resistance, which, according to James, is again not exactly what people think it is. "Our evidence implicates oxidative stress, and particularly that occurring in the mitochondria, as a very early step in the development of insulin resistance. In fact, we already have very revealing and quite neat results from experiments in animals to support this proposal."

As part of the second focus, the basic scientists in James's program have also just embarked on a large collaboration with hospital-based clinical researchers. The clinicians are recruiting individuals at risk of developing diabetes, but without any clinical signs of onset.

"Now armed with all the data and information and insights from our work in cells and animals models, we can now go into the human and ask if any of this stuff is relevant to the human condition. At the end of the day, the best model for what is going on with insulin resistance and diabetes is the human being, and we are very cognisant of that."

Cucumbers with warts

The third and newest area of the program is drug development. This is an expanding focus, and according to James, one of the flagship programs currently is the use of traditional Chinese medicines.

It started about four years ago with a visit from scientists of the Shanghai Institutes for Biological Sciences in China. They were looking for potential collaborative projects in the area of type 2 diabetes, a rapidly growing problem in China that correlates with its status as an emerging economy and the average body weight of the population.

It is estimated that by 2020, there will be as many Chinese suffering from type 2 diabetes as the total number in the world today. Sadly, the other economic powerhouse, India, is rapidly following suit.

"Our program seemed to fit quite well with what they wanted and it just went from strength to strength," James says. "In fact, we just had a paper accepted in one of the leading chemistry journals."

The work centres on a vegetable of the cucurbit family, which has been used to treat diabetes in China for several hundreds of years. It is called bitter melon (Momordica charantia) and is described by James as "looking a bit like a cucumber with warts".

"Our collaborators in China literally got hold of a tonne of the stuff, ground it up and started purifying all the small molecular components by high-throughput HPLC and other methods." The Chinese scientists solved many of the component structures to the atomic level, and many have turned out to be completely novel compounds - never before described.

The ones of most interest to James and colleagues fall into a chemical group called triterpenoids. The Garvan researchers tested a host of purified candidates using their established cellular assays and animal models of diabetes, and James was genuinely surprised at the results.

"Lo and behold, some of these compounds seemed to have insulin-like effects in our cells. Furthermore, when we administered them to animals, they had an insulin-sensitising effect. In other words, they improved the animal's glucose tolerance.

"This stuff really does blow me away. You start with a vegetable that has been used for eons to treat the disease in China, plus more than a little scepticism, and end up with new compounds that might one day be used to treat diabetes. It has turned out to be more exciting and promising than I ever would have imagined."

The importance of preventing or even delaying the onset of type 2 diabetes is clear simply from cost-benefits alone. Last year, Australia spent several billion dollars treating diabetes, which is fifth highest on the list in this regard. Delaying the onset of disease by 10 years, or even five years, could save the health system an enormous amount of money, not to mention dramatically improving the health of Australians.

The other interesting thing about the disease, according to James, is whether it is just a lifestyle disease - clearly a longer discussion than these pages allow, but it is one of the reasons that diabetes gets a bad rap. He and his colleagues at the Garvan have come to realise through their research that it is just not that simple. If it were, more would have it as our population ages and gets heavier by the mouthful.

As James says, "it is in a way a disease of progress and we could stop it if we could turn the clock back, but we can't. The desire to eat is very powerful. What coming back to the Garvan has emphasised for me is that at the end of the day we can't just sit in our labs and pretend it is just about curiosity. You have to constantly re-evaluate where you are going in terms of the human health perspective. It may be just a reflection of getting older, but I honestly believe that."

---PB--- Citation classic

David James is an internationally recognised expert in diabetes research. He has always been interested in the link between insulin signalling and glucose uptake into muscle and fat cells, a crucial step in the pathway to type 2 diabetes.

As a postdoctoral researcher with Paul Pilch at Boston University, and later with Mike Mueckler in St Louis, James identified and cloned the insulin-regulatable glucose transporter, GLUT4, which transports glucose from the blood into muscle and fat cells.

This work resulted in two Nature papers in quick succession (1988 and 1989), both of which are now citation classics (together cited over 1000 times). Since then, he has focused his attention on how insulin binding to its receptor at the membrane relates to increased glucose uptake. Along the way, he has identified several key steps in the insulin regulation of glucose transport, some of which are now major therapeutic targets in diabetes.

In 1999, James won the Glaxo Wellcome Medal in recognition of his significant scientific contributions. In 2006, the Australian Diabetes Society awarded James its highest accolade - the Kellion Award. Then, last year, he was made a Fellow of Australia's Academy of Sciences. In addition to being director of the diabetes and obesity program at the Garvan, James is an NHMRC senior principal research fellow.