Targeting the aspartic protease BACE1 for inhibition in order to increase hypothalamic leptin sensitivity and reverse obesity.

Chronic obesity is currently at epidemic levels in the UK and many other countries worldwide and is having a major adverse impact on our society. It is estimated that more 25% of the adult population are obese and around two thirds are either overweight or obese. Worryingly over the last 20 years there has been a ~3 fold increase in the number of children and adolescents who are obese or overweight. This trend is alarming, as chronic obesity is associated with increased risk of a number of diseases such as diabetes, cardiovascular disease, Alzheimer's and a number of cancers. It is clear that our society is on a trajectory that will see significant increases in the proportion of individuals entering or in old age with severe chronic health issues. This places an enormous burden on the NHS and society generally, which is likely to worsen over the next 10-20 years if effective actions are not taken soon.

The hormone, leptin, plays an important role in the maintenance of long term body weight and fat levels in animals and humans, It acts in the brain, in an area called the hypothalamus, to decrease feeding and increase energy expenditure. However, leptin's effects on appetite and energy expenditure are diminished in aged and obese individuals, despite increased circulating levels of the hormone. This phenomenon is known as leptin resistance and is thought to be due to reduced transport of leptin into the brain and/or a loss of the ability of leptin to alter the function of cells in the hypothalamus. Thus in non-obese individuals leptin alters the amount of certain neurotransmitters within neurons, their release onto nearby cells and their actions on these cells. These effects are lost or diminished through diet-induced obesity in both humans and animal models of obesity. Presently there are few effective drug treatments for obesity and none that overcome leptin resistance in the hypothalamus.

The enzyme, BACE1 cuts a larger protein known as amyloid precursor protein resulting in smaller protein fragments, some of which are thought to be toxic and responsible for the pathology associated with Alzheimer's Disease (AD). Indeed, excessive activity of BACE1 in the brain is thought to be the primary driver for the neurodegeneration and cognitive dysfunction associated with AD. The amount and activity of BACE1 increases with age and following pathological stresses such as hypoxia, oxidative stress (e.g. free radicals) and physical brain injury, thus increasing the risk of AD. Features common to AD and to diabetes and obesity are the loss of function of important metabolic hormones such as insulin and leptin and the reduced ability of cells to take up and use glucose.

Consequently, we hypothesised that BACE1 plays an important role that connects cell stresses (metabolic, oxidative and inflammatory) with glucose and fat metabolism. To date our work has shown that removing or decreasing BACE1 in mice causes them to be lean, resistant to weight (fat) gain on a high fat (calorie) diet and to improve their ability to deal with increased amounts of glucose in the blood (as occurs after a meal). In other words, these mice appear resistant to obesity and diabetes normally induced by chronic excess caloric intake. In a pilot study we show that by reducing the amount of BACE1, leptin is more effective in the hypothalamus and remains so even when animals become obese (i.e obesity is reversible by increasing the amount of leptin in the blood). This information suggests that reducing BACE1 prevents or reverses leptin resistance. Importantly our recent experiments show that giving obese mice (which are resistant to endogenous leptin) a drug that inhibits BACE1 causes significant weight loss. We now wish to confirm and extend these preliminary findings by studying a larger number of animals made obese by high fat diet and to try to define what has changed in the hypothalamus to circumvent leptin resistance.

Part 1: We will perform a comparative study of C57BL/6 BACE1-/-, BACE1+/-, and wild type littermate (BACE1+/+) mice, in which all three genotypes will be fed on regular or high fat diet for 20 (or longer for BACE1+/- mice) weeks (for HF diet sufficient time to induce DIO in wild type mice). In this longitudinal study we will determine how loss (BACE1-/- mice), reduction (BACE1+/- mice) and increased (via high fat diet) BACE1 affects RMR, body composition, food intake (basal and MTII modified) and plasma glucose and hormone (leptin, insulin) levels. At the end of the dietary period all three genotypes will be injected with leptin to explore the role of BACE1 on hypothalamic leptin sensitivity (measuring changes in food intake, hypothalamic neuropeptide expression and signalling strength of various leptin receptor-mediated pathways). These will be compared using peripheral and central routes of leptin administration.

Part 2: We will use the same criteria set out above to test the prediction that reduction of BACE1 activity in DIO wild type mice results in loss of body weight (reduced adiposity) concomitant with recovery of hypothalamic leptin sensitivity (i.e. reversal of high fat diet induced hypothalamic leptin resistance). Inhibition of BACE1 activity in mice will be induced through pharmacological inhibition by infusion or oral administration of small molecule non-peptide BACE1 inhibitors.

Part 3: We will determine whether the effects of BACE1 inhibition require the presence of leptin by oral administration of BACE1 inhibitor to ob/ob mice using the same output measures described above. Additionally, we will analyse dietary-modified leptin- and BACE1 inhibitor-treated hypothalamic tissue for evidence of BACE1-mediated alterations in signalling pathway networks associated with obesity and diabetes and/or known components of leptin signalling pathways. Peripheral tissues will be examined for evidence of altered levels of energy expenditure markers.

There is strong evidence to show that our modern (westernized) lifestyles have caused an epidemic of metabolic disease, notably through the large increase in diabetes associated with soaring levels of obesity. Indeed, around a quarter of all adults in the UK are obese, and over 60% are overweight or obese. Similar figures or trends are reported in other westernised countries. These changes are closely linked to lifestyle factors such as increasingly inactive occupations and daily routines and poor diet. Importantly chronic obesity has a severe impact on the health of individuals, increasing the risk of Type 2 diabetes (80% of T2D patients are obese), some cancers and liver and heart disease. Diabetes and obesity have also been shown to be significant risk factors in the development of forms of dementia such as Alzheimer's disease. Worryingly, the incidence of these diseases is expected to increase dramatically over the next 10 - 20 years (as we live longer), increasing the proportion of individuals entering or in old age with severe chronic health issues. Given this impact on long-term health this places a significant burden on the NHS (and similar health care providers elsewhere in the world). UK direct costs (medical, social and economic) are put at £16 billion per year. More alarmingly, this cost is predicted to rise steeply over the next 30-40 years, and is likely to seriously undermine the prosperity of current and future generations.

At present there is no long-term effective treatment for obesity, and none that have been demonstrated biologically to reverse the state of hypothalamic leptin resistance, which is thought to drive this steady-state increased level of body fat. In this context it is clear that alternative strategies to reverse/treat the obese state are urgently required and would be of great benefit to the UK population (medical, social and economic), by enhancing long-term health and improved quality of life for the individual. The preliminary studies performed in our laboratory provide strong support for a role for BACE1 inhibition in the reversal of the obese state in an animal model of diet-induced obesity. The current project aims to build on this exciting data and will directly test the hypothesis that hypothalamic BACE1 levels/activity markedly influences hypothalamic leptin sensitivity and susceptibility to obesity. Most excitingly, in recent years a number of companies have begun testing small molecule BACE1 inhibitors (for Alzheimer's disease treatment). Assuming our hypothesis is correct, a clear opportunity exists to intervene pharmacologically to inhibit BACE1 and reverse hypothalamic leptin resistance resulting in increased energy expenditure and reduced adiposity. Within our research collaboration grouping, clinical colleagues are well placed through running Diabetes outpatient clinics and through the Dundee Clinical Research Centre to conduct small-scale human trials with appropriate BACE1 inhibitors. As numerous BACE1 inhibitors are becoming available we expect a rapid translation of our experimental findings into human disease.

In summary, the work described herein may result in outcomes that:

(i) offer potential pharmacological-based treatments for obesity, which can be fast-tracked into small clinical trials, with the strong possibility that better drugs may be rapidly developed in association with Pharmaceutical companies.

(ii) enhance the quality of life (and should extend life) for a substantial proportion of the adult (and perhaps adolescent/child) population

(iii) improve the effectiveness of NHS care and treatment, reduced NHS and other governmental costs associated with obesity and its co-morbidities.