*BBSRC EASTBIO Programme*Lipid induced changes in mitochondrial function and mitophagy

Background:
Mitochondria are key structures within our cells that function as power plants, generating energy in the form of ATP from the “burning” of carbohydrate and fat within our diet. This energy powers numerous cellular processes including synthesis of macromolecules such as DNA and protein that are central to cell life. However, when mitochondria are exposed to sustained oversupply of nutrients, in particular to saturated fat, as seen during obesity, their capacity to efficiently “burn off” excess fuel is severely compromised as fuel supply greatly exceeds cellular energy demand. Under these circumstances, mitochondria become stressed and generate harmful molecules initiating their fragmentation and degradation by a process called mitophagy, which contributes significantly to the pathogenesis of metabolic diseases such as Type-II diabetes [1]. Strikingly, provision of mono or polyunsaturated lipids confers protection against mitochondrial dysfunction and ameliorates the deleterious effects of saturated fat [2]. Precisely how such benefits are accrued is poorly understood at present. This project will utilise cell and animal-based approaches to explore how mitophagy is regulated by fat provision at the molecular, cell and whole-body levels and provide novel insight and opportunities for developing nutritional and/or pharmacological strategies for treatment of obesity-related metabolic dysfunction.

Methodology:
Our working hypothesis is that excess fuel loading promotes mitochondrial dysfunction and mitochondrial clearance by mitophagy. To test this we will utilise cultured cells (muscle, heart and liver derived lines) treated for sustained periods with increased levels of saturated fatty acids and glucose (to mimic the obese/diabetic environment) in the absence/presence of mono or polyunsaturated fatty acids. Following this, cells will be exposed to fluorescent dyes (e.g. mitotracker, MitoSox Red, JC-1) whose mitochondrial accumulation (visualised by high resolution confocal microscopy) will inform how changes in fuel provision affects (i) mitochondrial morphology, (ii) production of mitochondrial reactive oxygen species and (iii) changes in mitochondrial membrane membrane integrity. Such measurements will be made alongside analysis of cellular oxygen consumption as readout of cell respiration and studies assessing how fuel provision affects the mitochondrial proteome. In parallel, we will also use cells expressing a tandem mCherry-GFP tag attached to the outer mitochondrial membrane localization signal of a protein called Fis1. Mitochondria in cells expressing this construct will fluoresce red and green. However, upon increased mitophagy, damaged mitochondria are delivered to lysosomes for removal where the low pH quenches GFP but not mCherry [3]. Consequently, these mitochondria form punctate structures and only fluoresce red allowing the degree of mitophagy to be quantified.

The project will also determine the impact of high fat feeding on mitophagy in skeletal muscle, liver and heart of mice in vivo. The Ganley lab has successfully engineered mice globally expressing the mCherry-GFP tag in their tissues which represents a valuable new research tool [3]. The project will determine the effect of placing mice on a control and high fat diet for 12 weeks, a regime that induces insulin resistance in tissues such as muscle and liver. In parallel, mice fed these diets will also be maintained on an exercise programme. At the end of this period, the student will (i) determine whole body insulin sensitivity and tissue responses to an acute insulin challenge, (ii) process thin tissue sections of muscle, liver and heart for microscopy to visualise mitophagy and (iii) perform mitochondrial isolation from these tissues for expression analysis of respiratory function, fusion and fission proteins. Collectively, the studies will provide, for the first time, an insight of how disturbances in metabolic capacity associated with increased dietary fat intake/obesity link to changes in mitochondrial content, structure and damage in key metabolic tissues and establish whether such disturbances can be mitigated by exercise.

Training offered:
A major objective of this project is to train a PhD student in both in vitro and in vivo methods and good research practice by performing a well-supervised, high-quality project at the forefront of international science in an intellectually stimulating research environment. The Hundal and Ganley groups occupy modern lab space and have full access to a number of core facilities (e.g. DNA/protein sequencing, oligonucleotide synthesis, microscopy, proteomics and an animal/transgenic unit). Our labs and those of our collaborators possess the basic equipment needed to perform the studies and resident post-doctoral/post graduate staff within our groups will provide a supportive and collegiate working atmosphere.