AMPK-mediated regulation and roles of miR-125b and miR-184 in pancreatic beta-cell function.

Diabetes affects 8% of the world population. When poorly treated, blood sugar levels run dangerously high, which can lead to blindness, kidney failure, limb loss and death. In the UK the NHS spends over £10 billion every year on diabetes care, but we are no closer to a cure. In fact, the incidence of type 2 diabetes (which accounts for >90% cases) continues to rise unabated. Central to the development of diabetes is the failure of beta-cells, which are dispersed throughout the pancreas in the islets of Langerhans. The failing beta-cells are unable to secrete enough insulin to lower blood sugar levels. Crucially, a high prevailing blood sugar will itself further accelerate beta-cell failure in a vicious cycle. A deeper understanding of the molecules that control beta-cell function and survival is essential for the development of better targeted drugs that can prevent, slow down or even reverse beta cell demise and hence effectively treat diabetes.
I have spent most of my career studying microRNAs (miRNAs), which are tiny molecules inside cells that have recently been discovered to regulate the genes that are required for a cell to function normally. There are more than 2000 different miRNAs in the human body and all cells, including the beta-cells, need miRNAs to work adequately. It has also been demonstrated that alteration of miRNAs can lead to diabetes. Nevertheless, very little is known about which miRNAs are important for beta-cell function, how they exert their influence and how their actions are controlled.
I have now demonstrated that two miRNAs, miR-184 and miR-125b, are closely related to the activity of AMPK. AMPK is an enzyme that helps the beta-cell recognize what the prevailing blood sugar level is and it is vital to their survival and normal functioning. Furthermore, I have demonstrated that these miRNAs themselves go up and down in response to changes in the amount of the sugar. All of this points to AMPK and its related miRNAs as being important potential drug targets for diabetes. In fact, existing diabetes medications are already thought to work in part by modulating AMPK, but our understanding of the exact mechanisms and molecular interactions is patchy, which limits the development of even more effective treatments.

In this project I will use state-of-the-art techniques to:
1. Fully elucidate the contribution of miR-184 and miR-125b (and their interactions with AMPK) towards beta-cell function and survival.
2. Show the real-life importance of this in animal models and donated human beta-cells - this is hugely important to ensure that this new knowledge can be potentially translated into highly effective new treatments for diabetes.

MiRNAs are small RNAs essential for endocrine function and altered in T2D. AMPK is an important energy sensor essential to maintain beta-cell function and survival by mechanisms not-well understood. My preliminary data show that miR-184 and miR-125b expression is altered in islets from mice lacking AMPK in beta-cells (AMPKdKO) and that beta-cell levels of both miRNAs are regulated by glucose. MiR-184 is already known to impact beta-cell function, whereas the role of miR-125b, a strong regulator of signalling and apoptosis in tumours, hasn't been studied in beta-cells.
I hypothesize that miR-125b impacts beta-cell function and survival, that AMPK mediates the effects of glucose on miR-184 and miR-125b expression in beta-cells and that miR-184/miR-125b in turn contribute to the effects of AMPK in beta-cell identity and function.
I will:
1. Assess the effect of AMPK depletion in the regulation of miR-184 and miR-125b by glucose in vivo and in vitro.
2. Use ChIP and reporter experiments to identify the regulatory sequences and proteins in the miRNAs' promoters.
3. Determine miR-184/miR-125b contribution to AMPK action by restoring the function of these miRNAs in AMPKdKO islets.
4. Apply the innovative iCLIP technique in beta-cell lines to identify miR-125b gene targets in a high-throughput manner.
5. Generate mice with genetic beta-cell-specific miR-125b alteration.
6. Transplant human islets with altered miR-125b levels into the mouse eye, generating a humanized mouse model with optically accessible islets to study in vivo.
7. I will use conventional and state-of-the art technologies, such as high-resolution in vivo confocal microscopy, to assess beta-cell function and survival.
This work will help to understand the mechanisms underlying AMPK and miRNA action in beta-cells while introducing the use of new cutting-edge technologies for the study of beta-cell function. This is vital for the development of better-targeted drugs to treat T2D.

Briefly, the research proposed here will:
(1) Contribute to understand AMPK mechanisms of action in beta-cells
(2) Identify new mechanisms underlying miRNA (miR-184/miR-125b) regulation and function
(3) Apply cutting edge techniques to the study of beta-cell dysfunction
Therefore, it will potentially benefit (see Pathways to impact for further details):
- Patients suffering from T2D
Diabetes incidence is rapidly increasing, affecting 422 million people worldwide (www.who.int/diabetes/en). Diabetes leads to serious complications such as heart attacks, stroke, kidney failure or vision loss with a strong economic impact to patients and health systems. In the UK, 3.8 million people suffer from diabetes, and its treatment cost the NHS over £10 billion per year (>10% of its full budget). Most importantly, 24000 people with diabetes die early each year in the UK (www.diabetes.org.uk). While T1D is a relatively well-characterized disease, T2D is a less defined condition in which defects in glucose sensing and loss of beta-cells combine to reduce insulin secretion in the face of insulin resistance, resulting in raised glucose levels. Understanding the mechanisms underlying the control of insulin secretion and the impact of currently used anti-diabetic drugs in beta-cell function and survival is essential for the development of much needed, new, more effective therapies and would have a huge impact in health and welfare of the UK population with T2D and in the UK economy.
- The biotechnology sector
Metformin is the most widely-used drug for the treatment of T2D. The mechanism of action of this drug is unclear, although AMPK is one of its probable sites of action. Moreover, AMPK activators might be useful in the prevention and/or treatment of cancer or heart disease. As such, several compounds including salicylate or phenobarbital have also been suggested to exert some of their beneficial effects though AMPK. Pharmaceutical companies such as Merck, Boehringer, Sanofi, Takeda or Astrazeneca have already spent millions of pounds to identify/develop more specific AMPK activators with fewer off-target effects for the treatment of those diseases and would strongly benefit of a better understanding of AMPK mechanism of action.
MiRNAs represent important potential therapeutic targets thanks to their small size, which makes them relatively accessible as chemicals or molecular targets, to their high conservation between species, which facilitates preclinical trials in animal models and to their ability to simultaneously target multiple targets within a molecular pathway. Not surprisingly, small biotech companies with a portfolio relying on miRNA-based therapeutics are now quickly emerging, such as miRagen, InteRNA or Regulus Therapeutics. As such, preclinical and clinical trials are being actively developed to target artheriosclerosis, heart failure or liver cancer. MiR-184 and miR-125b have been already suggested as therapeutic candidates for diabetes and certain types of cancer, respectively, but more information regarding their mechanism of action, regulation and function is essential.
- The Research and Education Sector.
This project will use state-of-the-art techniques that haven't been so far applied to the study of beta-cell function in the UK. These will benefit all UK scientist interested in the study of beta-cell function and diabetes, as well as other scientist that will be able to use protocols and materials generated during this study in their own fields.
The research assistant hired for this project will be trained in the latter techniques as well as in many more conventional but essential ones, acquiring a broad portfolio which will strongly advance his/her career. As part of the Section of Cell Biology and the Department of Medicine I will also be able to train and teach other students from the College. All the above will also boost my own career, by enhancing my technical, teaching, management and collaborative skills