The physiological role of ABCC5 (MRP5) in the regulation of hormone secretion from gut endocrine cells.

As modern humans, we have a complicated relationship with food and few among us would view food simply as an energy source. The preparation and consumption of food plays a central role in our daily lives and many of us can remember in great detail the tastes, smells and textures of our favourite childhood dishes. Furthermore, our brains have evolved to reward us for eating calorie-dense food because until very recently, starvation was a very real threat. However, currently we find ourselves surrounded by sugary, fatty foods 24/7, and in deep trouble.
Anyone who has ever been on a diet knows intuitively that appetite regulation is far more complex than a simple see-saw between feeling full and feeling hungry. A multitude of physiological signals regulate appetite and metabolism. Feelings of hunger is initiated by a shrunken, empty stomach through the vagus nerve, and the simultaneous secretion of the 'hunger hormone', ghrelin. These signals act on the brain to stimulate feelings of hunger. Feelings of satiety are signalled to the brain shortly after food arrives in the stomach; a distended stomach and a sharp drop in ghrelin levels mute hunger signals to the brain and the arrival of digested food in the small intestine engages a large array of hormone secreting cells, known as enteroendocrine cells. Hormones secreted from enteroendocrine cells regulate the secretion of digestive enzymes from the gall bladder and pancreas, the rate of movement of food through the gut, the secretion of insulin from the pancreas and feelings of satiety in the brain. Gut hormones therefore have many down-stream targets and act on the vagus nerve which innervate the gut, the brain, the distant hormone-secreting cells of the pancreas, and also on their enteroendocrine neighbours. This constant chit-chat between the hormone secreting cells of the gut and their targets is the focus of my research.
Digested food is detected by receptors expressed on the membranes of enteroedocrine cells. Activation of these receptors trigger a cascade of events that culminates in the secretion of hormones from endetroendocrine cells, a process called exocytosis. Conversely, there should also be a brake in place, to turn exocytosis off once adequate hormone had been released. The detection of nutrients in the gut have been well studied, but we currently know little about the sequence of events which terminate hormone release and the communication taking place at the junctions where enteroendocrine cells and nerve endings colocalize. Specifically, my research investigates the role of a membrane transporter, ABCC5, which has been shown to transport neurotransmitters across cell membranes. This protein appears to play a central role in turning exocytosis signals off. When ABCC5 protein levels is artificially boosted by recombinant overexpression in a model cell line, hormone release is inhibited. When ABCC5 protein levels are knocked out, exocytosis is enhanced. In addition, an ABCC5 substrate, a neurotransmitter called NAAG, also inhibits exocytosis. Taken together, this would suggest that ABCC5 and molecules it transports across the enteroendocrine cell membrane is involved in switching exocytosis off.
Our research investigates the role of enteroendocrine cells from two angles. First, we aim to identify the receptors and enzymes involved in the signalling cascades which regulate and terminate hormone exocytosis. Secondly, we take a wider whole-systems look and investigate the metabolic implications for knock-out mice, which lack ABCC5. If our hypothesis holds true, mice lacking ABCC5 will secrete more hormone in response to the arrival of digested food in the small intestine, will have a more robust insulin response and will display a 'lean phenotype'. Our research will help us understand the whole body impact of gut hormones and will help scientist from several different areas to make connections between systems previously studied in isolation.

Endocrine L cells are specialized nutrient sensing cells in the small intestine and release hormones, such as GLP-1, in response to digested food which regulate appetite and insulin release. Reversal of pathological insulin resistance observed immediately post bariatric surgery is correlated with enhanced hormone release from gut endocrine cells. This enhanced endocrine response is the result of digested food delivered to more distal parts of the small intestine. Autocrine and paracrine feedback loops which regulate hormone exocytosis are well established for non-gut endocrine cells like pancreatic alpha and beta cells, but little is known about regulation of exocytosis events of gut endocrine cells.
Preliminary data from our laboratory using a model L cell line, GLUTag cells, suggests a role of a membrane transporter, ABCC5, in negative feedback regulation of L cell activity: decreased ABCC5 expression leads to enhanced GLP-1 release, while overexpression of this transporter attenuates GLP-1 exocytosis. Notably, polymorphisms in the exporter ABCC5 have been linked to diabetes in humans and ABCC5 overexpression is associated with reduced peripheral insulin sensitivity in nondiabetic individuals, increased visceral fat accumulation and increased risk of developing Type 2 Diabetes. The proposed study will investigate (1) the molecular mechanism of ABCC5-dependent regulation of hormone exocytosis from L cells and (2) the role of ABCC5 in metabolic homeostasis using ABCC5 knock-out mice. Specifically, the study will aim to identify the downstream receptor targets and signal transduction pathways involved in the regulation of ABCC5 dependent hormone exocytosis in L cells, and will investigate the metabolic effects of diet on knock-out mice. Understanding how L cells initiate and terminate exocytosis events in response to nutrients will make a significant contribution to understanding the complex mechanisms underpinning metabolic disorders such as diabetes and obesity.

The potential long-term impact of this research for the public and the economy is substantial. The current obesity epidemic and the unsustainable drain on medical resources by pathologies associated with a Body Mass Index (BMI) of 30-35 kg/m2 is well documented. Obese patients have a 60% increased risks of developing type 2 diabetes (T2DM), 20 % increased risk of hypertension and coronary-heart disease (heart attacks and stroke) and are significantly predisposed to cancers of gall bladder, kidney, ovaries and pancreas as well as chronic respiratory diseases (such as chronic obstructed pulmonary disease and asthma). In 2010, obesity overtook smoking as the major cause of morbidity, disability and premature death.
Statistics released in 2014 indicated that 64% of Britons are overweight and 1 in 3 middle-aged adults can be classified as obese. Babies born to overweight mothers have a 3-fold higher chance of being overweight and 77% of obese children become obese adults. The average obese person costs 36% more in medical care and in the UK, healthcare costs attributable to increased BMI is projected to be £10 billion per year, with the wider economic costs estimated to reach £50 billion per year by 2050. A study in 2010 showed that 200 million school children world-wide can be classified as overweight, 40 million of these children were under the age of 5 and the youngest child diagnosed with lifestyle-induced T2DM, is a 3 year old toddler weighing 35 kg (77 pounds).
At present, bariatric surgery remains the only treatment guaranteed to result in sustained, long term weight-loss in morbidly obese patients. This weight loss is attributed, in part, to altered physiology of gut hormones and their appetite-regulation capabilities. But, this invasive treatment is not a sustainable solution for a large population and is ethically questionable in young children. There is therefore an urgent need to elucidate the mechanisms which drive stimulus-secretion coupling of hormones in the gut if we aim to develop new ways to treat obesity with a non-invasive, non-surgical approach.
ABCC5 overexpression in humans is significantly associated with reduced peripheral insulin sensitivity in nondiabetic individuals with an associated increased visceral fat accumulation and increased risk of developing T2D with age [6]. This trend was observed in both European and African American population studies and included data from 3 populations, the Wellcome Trust Case Control Consortium (T2D cases: n=1926; controls: n=2938), the African American National Institute for Diabetes and Digestive and Kidney disease (T2D cases: n=1033; controls: n=971), the European twins consortium (T2D cases: n=256). Even though this result strongly suggests that ABCC5 plays an important role in human metabolism, the role of ABCC5 in diabetes and obesity remains largely unexplored.
We are just beginning to understand how hormone exocytosis is initiated in gut endocrine cells, but little is known about the signals that terminate hormone release. Research into understanding the basic mechanisms underpinning stimulus-secretion coupling in gut endocrine cells could give us valuable insight into the deregulation of metabolic- and signal transduction pathways in these cells in the overweight and obese. Research which contributes to a better understanding of why our satiation signals go awry, and the molecular mechanisms which regulate the synthesis and release of gut hormones pivotal in these signalling pathways, will have a major impact on addressing the pressing medical issue of human obesity.