MICA: Deciphering the mechanism of action of miR-125b in beta cells and its therapeutic potential in Diabetes

Over 8.5% of the world's adult population suffer diabetes. If poorly treated, diabetes leads to very high blood sugar levels which worsen the disease and lead to complications such as kidney failure and blindness, shortening life expectancy by 10 years in the case of type 2 diabetes (T2D).
Pancreatic beta cells are in charge of secreting insulin in response to rises in blood sugar. Failure of beta cells to secrete enough insulin contributes to the development of diabetes. Importantly, the prevalence of high-blood sugar accelerates beta cell failure and contributes to beta cell loss by mechanisms which are not yet clear. A better understanding of the process leading to beta cell failure is vital for the development of drugs capable of stopping the development of T2D.

MiRNAs are small RNA molecules that do not produce proteins themselves but are capable to reduce the rate at which other proteins (their "targets") are produced. MicroRNAs exist in beta cells that regulate important functions such as their capacity to produce and secrete insulin. Also, changes in the levels of certain miRNAs in beta cells are associated with the development of T2D.

We have recently made three important findings. Firstly, when mouse and human beta cells are exposed to high levels of glucose, their levels of the miRNA miR-125b (miR-125b-5p) go up. Secondly, the introduction of additional miR-125b in the beta cells of mice causes them to produce and secrete less insulin and develop diabetes. We have also observed that reducing the amount of miR-125b in human beta cells in culture improves their capacity to secrete insulin in response to glucose. Accordingly, we hypothesize that beta cell selective inhibition of miR-125b has the potential to protect beta cell function from hyperglycaemia. Thirdly, we have seen that high levels of miR-125b lead to the appearance of enlarged lysosomes while low levels of miR-125b lead to changes in mitochondria morphology and in the content of genes related to mitochondrial function. Lysosomes and mitochondria are subcellular organelles very important for the recycling of cellular components and waste and for energy production, respectively. Thus, we hypothesize that miR-125b regulates beta cell function by modulating lysosomal and/or mitochondrial function. Both processes are essential for adequate beta cell function and are altered in diabetes.
Additionally, we have demonstrated that miR-125b targets the cation-dependent lysosomal mannose-6-phosphate receptor (M6PR) which transports lysosomal enzymes to lysosomes for their adequate functioning. Nevertheless, the role of M6PR for lysosomal and secretory function in beta cells hasn't been studied.

Thus, the specific aims of this proposal are to determine:
1. Whether and how selective elimination/reduction of miR-125b in beta cells prevents T2D progression
2. The role of miR-125b in lysosomal and mitochondrial function
3. The function of M6PR in beta cells

To achieve these aims we will use a combination of
- Mice deleted for/overexpressing miR-125b selectively in beta cells. The use of mice is necessary since maintenance of glucose homeostasis requires interplay between all metabolic tissues and therefore these experiments need to be done in the context of the whole body.
- Donated human islets, modified to contain more or less miR-125b. The use of human samples is essential to ensure the translatability of our findings to the clinic.
- Mouse and human beta cell lines, modified to contain more or less miR-125b or M6PR, which allow to study biological processes in detail and reduces an unnecessary use of animals.

MiRNAs are novel candidates for drug targeting and our study will provide preclinical data on the potential of beta cell miR-125b inhibition for the treatment of T2D. It will also provide new fundamental insights into how beta cells work in health and disease, which, in the long term, could reveal new ways to treat diabetes.

MiRNAs are small RNAs that silence gene expression, essential for endocrine function. We have shown that islet miR-125b levels correlate with BMI and are increased by high glucose via AMPK. MiR-125b deletion in human beta cells improves insulin secretion and, conversely, beta cell selective overexpression of miR-125b impairs glucose tolerance and insulin production and secretion in mice. MiR-125b targets lysosomal and mitochondrial proteins (including M6PR, a transporter of lysosomal hydrolases) and its modulation affects lysosomal and mitochondrial morphology.
Thus, we hypothesize that miR-125b regulates mitochondrial and lysosomal function and that beta cell selective inhibition of miR-125b will improve beta cell function and glycaemic outcomes. We will:

1-Test whether miR-125b inhibition in beta cells improves secretory function in vivo, genetically eliminating miR-125b from mouse beta cells, using novel miR-125b inhibitors targeting beta cells and generating "humanised" mice by transplanting human islets with reduced miR-125b levels into the mouse eye

2-Dissect the role of miR-125b in lysosomal and mitochondrial function and its contribution to glucose/AMPK-mediated regulation of these processes, using cell lines and islets with miR-125b gain/loss-of-function

3-Determine the role of M6PR and the impact of its regulation by miR-125 in beta cells, using CRISPR/Cas9 and lentiviral vectors in islets and cell lines

We will use conventional and state-of-the-art technologies, such as high-resolution microscopy and molecular sensors to functionally characterize these models.

This work will unravel the mechanism of action of miR-125b in beta cells and demonstrate its therapeutic value for T2D treatment. We will generate new insights into the regulation of lysosomal and mitochondrial processes, increasing the understanding of the mechanisms contributing to beta cell function and failure, key to develop better therapies for diabetes in the future.