RAMP2 and the glucagon receptor: important in the pathogenesis of non-alcoholic fatty liver disease?

Background:

Non-alcoholic fatty liver disease (NAFLD or 'fatty liver') is a serious modern health problem. Its incidence is dramatically rising, and an estimated 1 in 3 British adults are now affected. It is a silent killer since initially it has no symptoms, but it can progress to liver failure and liver cancer. At present there are no specific treatments and the way it develops over time is poorly understood.

NAFLD occurs when too much fat is stored in liver cells. The hormone glucagon decreases fat storage in liver cells. RAMP2 is a member of a family of proteins (receptor-activity-modifying-proteins); and it is known to interact with the glucagon receptor. If we understand how RAMP2 changes the way glucagon works, this would give us an opportunity to design drugs that treat NAFLD.

My research to date:

I have studied the effect of RAMP2 on glucagon activity in liver cells. I have shown for the first time that increasing the amount of RAMP2 in liver cells decreases the effect of glucagon. On the contrary, reducing the amount of RAMP2 increases the effect of glucagon. Since RAMP2 decreases the action of glucagon, a hormone which prevents excessive fat storage, I hypothesised that it might contribute to development of NAFLD. To model this in living liver cells, I exposed them to a fatty acid called palmitate, which is raised in the blood of people with NAFLD. I found that palmitate exposure increases RAMP2 gene expression in these liver cells, which as expected, decreases the effect of glucagon.

Furthermore, I also examined liver tissue from mice. I have found that RAMP2 is increased in mice with fatty liver due to a high sugar diet.

These findings are very exciting, because they strongly suggest that RAMP2 may be involved in the development of NAFLD and that ultimately therefore it may be a new target for drugs to treat or prevent NAFLD.

My main hypotheses are as follows:

1. Increased RAMP2 decreases glucagon action in the liver
2. Increased liver RAMP2 contributes to the development of NAFLD

Aims of this study:
1. To confirm that fatty liver is associated with increased liver RAMP2
2. To see whether increasing RAMP2 worsens development of fatty liver in a mouse model
3. To describe in detail the effect of RAMP2 on the function of the glucagon receptor in liver cells

Key methods:

I will feed different diets to groups of mice, to see the effect on liver RAMP2. I will also study the effect of increasing the amount of RAMP2 made by liver cells, to see whether this increases the development of fatty liver. Although mice are a good model of human disease, I will confirm that the RAMP2-glucagon receptor relationship is important in humans as well by measuring the amount of RAMP2 in patients with normal or fatty liver disease.

My experiments using mice have been designed to use the minimum number to allow important scientific findings, and in the most humane way possible. In my experiments using human liver tissue I will use biopsies that are being taken anyway as part of clinical investigations.

If successful, my project will prove that RAMP2 is of key importance to the development of NAFLD, and also provide information as to how RAMP2 has its effects. This would be a huge step forward in understanding the important problem of NAFLD, and would open the door to development of specific drugs to prevent and treat it.

Evidence suggests that reduced hepatic glucagon action may contribute to development of non-alcoholic fatty liver disease (NAFLD), a condition that affects 1 in 3 British adults. Receptor activity-modifying protein-2 (RAMP2) interacts with the glucagon receptor (GCGR) and affects its ligand binding and intracellular signaling. To date this interaction has not been studied in hepatocytes.

My pilot data demonstrates that RAMP2 upregulation decreases activity of glucagon in Huh7 hepatoma cells. Further, I have shown that RAMP2 expression is dramatically increased in a mouse model of NAFLD. Additionally, in Huh7 cells upregulation of RAMP2 occurs in response to palmitate exposure, a fatty acid increased in the circulation of patients with metabolic syndrome. This data supports my theory that upregulation of RAMP2, via its effects on the glucagon receptor, is important in the pathogenesis of NAFLD.

My experiments will establish the effect of upregulated hepatic RAMP2 on lipid metabolism in mice and determine if increased hepatic RAMP2 expression contributes to dietary-induced steatosis. I will use recombinant adeno-associated virus to specifically upregulate RAMP2 in hepatocytes of adult mice. Furthermore, I will employ histological techniques to establish if the association between RAMP2 and steatosis holds in humans. Alongside, I will investigate the cellular basis of the effects of RAMP2 on the glucagon receptor. This will include use of an established protocol to investigate trafficking of the GCGR using a SNAP-tag in conjunction with confocal microscopy and flow cytometry.

This project will dramatically enhance understanding of the pathogenesis of a very common and serious medical condition, with the potential for therapeutic exploitation. More generally, this project will expand current understanding of receptor-interacting proteins, with far-reaching applications for science in related and diverse fields.

This project aims to show that downregulation of glucagon-induced hepatic lipolysis by RAMP2 is material to the pathogenesis of NAFLD. If true, this will open new therapeutic avenues for NAFLD, a condition which is estimated to affect one-third of British adults.

Since NAFLD is a huge and escalating health problem, this research has long-term potential to benefit a vast number of patients. The development of effective therapies to treat or reverse NAFLD will be cost-saving to society as a whole, as NAFLD is now a leading cause of liver transplantation world-wide.

If my work confirms that upregulation of RAMP2 contributes to the development of NAFLD, this would lead to the development of drugs that target either hepatic RAMP2 or the RAMP2-glucagon receptor interaction. My department has an impressive track record of translation of basic science to clinically important medications, for example gut hormones (e.g. oxyntomodulin) and their analogues (e.g. GLP-1 analogues). The development of new pharmaceutical agents for NAFLD would have considerable economic benefits, both for Imperial College and the pharmaceutical industry in the UK, especially given the prevalence of the condition.

If my research confirms that in animal models RAMP2 is differentially upregulated by diet, and that it contributes to the development of NAFLD, it will be important in future work to ascertain if this is the case in humans. Researchers in other fields such as nutrition and epidemiology would have opportunities to carry out related work to further define the relationship between diet, RAMP2 and NAFLD. Information generated would be of significant interest to public health policy-makers, who would use it to improve the effectiveness of their policies regarding healthy eating. The information would also be used to guide predictions of NAFLD national prevalence, based on current eating habits, which would enable better health care planning.

In the short-term, my experimental methods and findings will be of particular interest to other groups looking at RAMP2 and the glucagon receptor in other cell types, who will want to determine if RAMP2 has similar effects on glucagon action in different cellular environments (e.g. the pancreas). More widely, defining the importance of RAMP2 in this context is likely to spark further interest in receptor modifying proteins in other contexts. RAMPs are known to interact with several other receptors which to date have not been fully explored. Some of the experimental protocols which I will develop could be replicated by scientists looking at different RAMP-receptor interactions. This could lead to better understanding of the pathogenesis and potentially new therapies for a host of diverse conditions. For example, RAMP1 interaction with the calcitonin-like-receptor has already been exploited and has led to the development of small molecule drugs for migraine which are in clinical trials.

This project is therefore of considerable potential benefit to patients with NAFLD and other health problems, policy makers and society as a whole, and the wider research community.