REGULATION OF HUMAN GLUCOSE HOMEOSTASIS BY THE NOVEL CHC22 CLATHRIN ISOFORM

Type 2 Diabetes (T2D) and Insulin Resistance (IR), which result in excessively high blood sugar (glucose), generate major health problems affecting 382 million people worldwide and ~6% of the UK population, creating an enormous economic burden on modern society. Development of effective therapies is imperative, and requires fundamental research to identify new therapeutic targets and improve our understanding of human glucose metabolism. This research grant focuses on CHC22 clathrin, a novel regulator of human glucose transport, with potential to influence the health and wellbeing of people living with T2D and IR.

Proteins are molecular machines inside cells, and like any machine, a protein must be at the right place at the right time to perform its function properly. Clathrins are proteins responsible for transporting other proteins from one part of the cell to another, a process known as intracellular trafficking. The proposed research investigates a form of clathrin called CHC22 that we have found plays a role in intracellular trafficking of the GLUT4 glucose transporter protein, which regulates blood glucose levels.

After a meal, insulin is secreted from the pancreas. In response, glucose is imported from the blood into muscle and fat by GLUT4, a channel through which glucose can pass. During fasting, GLUT4 is held inside cells in the GLUT4 storage compartment (GSC). GLUT4 is released from the GSC to the cell surface in response to insulin produced after feeding, allowing glucose uptake and clearance from blood. In IR, tissues stop releasing GLUT4 and importing glucose in response to insulin, and eventually the pancreas stops secreting insulin (T2D). We have observed that, in muscle from T2D patients, when GLUT4 does not get to the surface after insulin stimulation, GLUT4 is trapped in the GSC together with excessive amounts of our protein of interest, CHC22 clathrin. We hypothesize that the presence of CHC22 at the non-functional GSC contributes to IR.

The most familiar role of clathrin is to move proteins from the cell surface to the inside of the cell, known as endocytosis. Multiple molecules of clathrin assemble to form a coat on the inside surface of the cell. The coat pulls the membrane to the inside and eventually the membrane breaks away, forming a coated structure that takes cell-surface proteins with it and carries this cargo to specific intracellular locations. In humans, there are two types of clathrin. Unlike the common CHC17 form, CHC22 is not involved in endocytosis, but has a specialized role in transporting GLUT4 to the GSC. We will characterize this role by analyzing the molecular and cellular properties of CHC22 and how its function is controlled by other proteins.

Proposed experiments will define how and where formation of the CHC22 clathrin coat is regulated in cells and characterise CHC22 behaviour changes in response to insulin and and IR. We will also explore the differences between two forms of CHC22 proteins found in humans - differences that may influence the development of IR. Together, these studies will reveal the molecular control of CHC22 function in human glucose regulation.

Scientists studying GLUT4 have not yet fully defined properties and regulation of the human GSC, and our experiments will shed light on this fundamental aspect of human nutrition. We will also develop new tools that can be used by other researchers studying IR and T2D. Our studies will further clarify the field of clathrin biology, which has been primarily focused on the roles of CHC17.

This research addresses how membrane traffic of the GLUT4 glucose transporter in humans is regulated by CHC22 clathrin. Insulin-stimulated delivery of GLUT4 to the cell surface from an intracellular GLUT4 storage compartment (GSC) is the primary mechanism for post-prandial glucose clearance from blood into muscle and fat. We discovered that CHC22 clathrin mediates formation of the human GSC, and accumulates at the GSC in muscle of Type 2 diabetes patients, when GLUT4 is trapped inside due to insulin resistance (IR). CHC22 clathrin is a novel isoform of clathrin that functions in a distinct pathway from the familiar clathrin (CHC17) involved in endocytosis.

This research grant will define CHC22 function and regulation with three aims.
- Advanced imaging of human muscle cells will produce a comprehensive map of CHC22 membrane traffic within the GLUT4 pathway. The molecular mechanism of changes in CHC22 traffic following insulin treatment and in models of IR in human muscle cells will be addressed through analysis of phosphorylation and changes in CHC22 partner proteins.
- Proteomics and functional assays in human myotubes will be used to discover proteins involved in CHC22 regulation under different nutritional conditions. Classical biochemistry will be used to define the mechanism of CHC22 uncoating from membranes and to compare the assembly properties of common genetic variants of CHC22.
- CHC22 genotyping will be performed from DNA of human subjects participating in studies of whole-body glucose control (Univ of Bath) to correlate genetic variation with measured parameters of glucose homeostasis. CHC22 transgenic mice will be produced for breeding and nutrition experiments (Univ of Oklahoma) to assess the influence of CHC22 variation on development of IR in mice expressing human GLUT4.

These studies will impact the cell biology and diabetes fields by addressing the molecular basis for CHC22 regulation of human glucose metabolism during health and disease

Type 2 Diabetes (T2D) is a major health problem, affecting 3.7 million people in the UK (~6% of the UK population) and estimated to afflict 8.8% of the global adult population in 2017. About 10% of the NHS budget is spent on T2D and related complications. A significant cause of T2D is insulin resistance (IR) resulting from obesity. The development of new and effective therapies for T2D and IR is imperative, and will require novel fundamental research to identify new therapeutic targets and to improve our understanding of human glucose homeostasis.

Who will benefit from this work and how?

Academic colleagues: New information about membrane traffic pathways that specifically affect human glucose homoeostasis will be generated by the proposed studies that are complementary to prevailing cell biology studies in murine cells. By elucidating molecular mechanisms for diversification of membrane traffic in specialized tissues, this research programme will establish connections between basic cell biology pathways and differentiated physiological functions relevant to metabolism.

Project staff: New research skills in biochemical and biological techniques will be acquired, enabling scientific career development. The focus of this research on human nutrition will encourage an outlook on public health and wellbeing beyond engagement in academic cell biology. Staff will participate in local and international symposia and conferences discussing the clinical impact of metabolic disease, as well as social factors influencing metabolic disease. Such participation will provide them with versatile presentation, communication, and professional skills. By contributing to the training and development of project staff, and by promoting a culture of excellence, we will enhance the global competitiveness and impact of UK scientific research.

Local clinicians and epidemiologists: Our participation in the local London metabolism community through Food, Metabolism and Society (FMS) @ UCL will introduce these beneficiaries to features of human biology that should be considered for impact on disease and nutritional management. Considering novel aspects of the biology and genetics of glucose metabolism may stimulate new genetic approaches to patient and population analysis. Of potential interest to clinicians, information gained about differential function of CHC22 genetic variants could have relevance to genetic predisposition to IR and T2D that might be nutritionally regulated to prevent onset. This engagement and the recent successful funding to launch FMS @ UCL are described in the Pathways to Impact statement.

Pharmaceutical industry: Identifying CHC22 clathrin as a key player in GLUT4 retention and storage suggests that the CHC22 pathway is a potential target for therapeutic treatment of insulin resistance. In the medium term, it is conceivable that our work could lead to novel therapeutic strategies for interrupting pathways to insulin resistance.

Patients with IR and T2D: By focusing on the cell biology of the GLUT4 pathway in humans, our work will, in the long term, impact the health and wellbeing of millions of people living with T2D and IR. Our research could lead to translational studies, drug development, and personalized medicine approaches that will ultimately increase the health and quality of life of these patients. Outreach activities planned through FMS @ UCL will further aid in educating this community about our work and its potential outcomes.

In conclusion, our work will have an impact on, and beyond, the academic disciplines of GLUT4 trafficking and clathrin cell biology, extending to clinicians and the general public. In the Pathways to Impact section we detail our plan to ensure these impacts are achieved.