Establishing AMP-activated protein kinase as a regulator of adipose stem cell plasticity and function in health and disease

To grow, replicate and function, cells must sense and respond to their environment. Energy, in the form of adenosine triphosphate (ATP) is required for all cellular processes; to build proteins, replicate DNA, repair membranes and fight infections. To maintain optimal ATP concentrations, multi-protein systems continuously monitor nucleotide levels within the cell, driving growth and proliferation when ATP is plentiful, and by halting energy expenditure and initiating catabolism when ATP is scarce. One such network is governed by AMP-activated protein kinase (AMPK), an enzyme found in almost all organisms, responsible for directly sensing cellular energy levels. Activation of AMPK under energy stress drives the breakdown of energy stores and the removal of damaged or malfunctioning mitochondria (mitophagy), where the majority of ATP is generated. AMPK activation also increases the production of new, healthy mitochondria (biogenesis), leading to efficient ATP production, and encourages the uptake of glucose by the cell, to restore and maintain ATP. AMPK therefore presents an attractive drug target for the treatment of many diseases in which alterations in energy homeostasis feature prominently.
Stem cells reside in most tissues, and are responsible for tissue maintenance and repair after damage. Metabolic diseases, such as obesity, diabetes and cardiovascular disease, often occur when energy balance is dysregulated, and are difficult to reverse with lifestyle change alone. Under conditions of metabolic stress, such as inflammation, hypoxia and cell exhaustion, these stem cells do not respond appropriately and the damage becomes irreversible. This process is also true of aging, when stem cells that reach their limits of self-renewal are no longer capable of tissue repair.
White adipose tissue, a specialised organ primarily responsible for hormone production and the safe storage of lipids, is often implicated in metabolic disease. Excessive expansion of white adipose tissue leads to adipocyte death, systemic inflammation and insulin resistance, hallmarks of metabolic syndrome. Adipose-derived stem cells (ADSCs) share many features of pluripotent stem cells, capable of forming a diverse range of cell types, including cartilage, bone, skeletal and cardiac muscle. The combination of accessibility and plasticity make ADSCs attractive for targeted stem cell therapies in regenerative and restorative medicine.
Our previous work identified a novel population of adipocytes, which we termed SMART cells, generated by genetically activating AMPK in a mouse model of obesity. These cells have a high mitochondrial content, increased mitophagy and express proteins usually found in muscle that allow them to generate heat, protecting the mice from diet-induced obesity and associated organ damage. As SMART cells express these muscle-specific proteins when stimulated in culture, they may be useful for the treatment of metabolic and age-related disease. However, little is known about the origin of these cells, or why they develop instead of normal white adipocytes when AMPK is active.
Our aim is to understand the role of AMPK in the development of normal human adipose tissue, and the consequence of metabolic alterations at key points in stem cell differentiation on cell fate and function. Using human immortalised adipocyte derived stem cells (h-iADSCs), and fresh human ADSCs, we aim to establish the role of AMPK in adipose stem cell maintenance, in the specification of cell lineage, and in the resulting function of the mature adipocyte. The work will also explore the effect of metabolic instability on adipocyte lineage and function by isolating ADSCs from patients with metabolic disease. To determine the therapeutic value of AMPK activation, we will transplant patient-derived ADSCs, treated with AMPK activator C455, into mice, where we will assess their ability to form whole adipose tissue, and their effect on whole-body energy homeostasis.

Targeting key regulatory networks governing stem cell fate for the treatment of metabolic and age-related diseases relies on a deeper understanding of the link between cell lineage and function. This project aims to identify the role of AMP-activated protein kinase (AMPK), an evolutionarily conserved heterotrimeric serine/threonine kinase, and its downstream effectors in transcriptional and metabolic regulation of human adipose derived stem cell (ADSC) diversity, lineage commitment and energy expenditure. Human immortalised ADSCs will be used as a model of adipogenesis, and commitment to lineage, in the presence/absence of small molecule AMPK activator C455. Metabolomic and transcriptomic (ChIP-sequencing) analysis at developmental checkpoints will monitor the influence of AMPK activation on TCA-cycle intermediates, and on transcriptional changes associated with specification to lineage. Functional readouts including fixed and live imaging, mitochondrial function/energy expenditure analysis, and substrate utilisation will assess the impact of AMPK and associated metabolic regulators on terminal differentiation and function under environmental stress, including hypoxia, beta- adrenergic stimulation, nutrient overload/deprivation and mitochondrial damage. ADSCs from both healthy volunteers and patients with metabolic disease will be treated with C455 ex vivo, and monitor as previously described. Responsive cells will be injected subcutaneously into immunocompromised mice to establish adipogenic potential in vivo, and to determine whether they convey a protective metabolic effect in vivo. Overall, this work provide the first evidence of whether targeting AMPK in human ADSCs has therapeutic potential. In addition, I will establish s a metabolomic-transcriptomic map of human adipocyte differentiation in health and disease.