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Report

Teaser, summary, work performed and final results

Periodic Reporting for period 1 - EXNADMINA (EXercise as a regulator of hepatic NAD metabolism and MItochondrial function in Non-Alcoholic fatty liver disease)

Teaser

Summary

Non-alcoholic fatty liver disease, a disorder which is associated with obesity, has become the most prevalent liver disease in industrialized countries, including Europe. Fatty liver disease comprises a spectrum of disorders ranging from hepatic fat accumulation (steatosis) to inflammation (non-alcoholic steatohepatitis) and fibrosis. In approx. 10% of patients this in turn leads to liver failure or the development of liver cancer (hepatocellular carcinoma) later in life. Apart from severely restricting personal health and wellbeing, the later stages of fatty liver disease also lead to substantially increased healthcare-related costs.
The development of fatty liver disease and other metabolic disorders has been associated with a decline in cellular levels of NAD which is both an essential cofactor for cellular energy production from different nutrients and a substrate of enzymes that regulate energy metabolism. Experiments with mice with obesity and fatty liver disease induced by a diet high in fat show that supplementing NAD using NAD precursors which belong to the vitamin B3 group has beneficial effects on cellular metabolism and can counteract the negative effects of a high fat diet.
Cellular organelles called mitochondria are mainly responsible for cellular energy production and are dependent on the correct levels of NAD and its derivative NADP. An enzyme which is essential in regulating mitochondrial NAD and NADP termed Nnt (nicotinamide nucleotide transhydrogenase) does not function in commonly used laboratory mice due to a mutation. These mice show defects in mitochondrial function but have been used almost exclusively in studies examining the effects of NAD supplementation in obesity and fatty liver disease. We hypothesized that mice having this non-functional version of Nnt will respond differently to a high fat diet and NAD supplementation compared to mice with a functional version of this enzyme.

The aim of this project was to evaluate these two different, but commonly used mouse strains (with and without functional Nnt). By addressing this fundamental question we aimed to provide knowledge useful for the interpretation of mouse experiments in the field of obesity research and associated diseases and at the same time shed light on the manner how obesity and fatty liver disease develop and which mechanisms are involved.

Work performed

The first period of the fellowship was used to establish all necessary methods for the measurement of mitochondrial function on cells in which a mitochondrial malfunction was induced. This included the state of the art high resolution respirometry for measurement of mitochondrial oxygen consumption. For inducing a dysfunction in mitochondria, both a pharmacologic approach applying a chemotherapeutic agent and a genetic approach (downregulation of the Nnt enzyme) were used. When using the chemotherapeutic agent, we found that, although NAD levels declined in the cells treated with this agent and cells died subsequently, the cells did not survive better if supplemented with NAD. This resulted in a collaboration of the fellow with researchers from Leipzig University, Germany, where the fellow is now working and continuing the collaboration.
In the second part, two different mouse strains were challenged with a high fat diet to induce metabolic stress, obesity and fat deposition in the liver. These mice either received NAD supplementation or not. In this experiment the method of supplementation of NAD via drinking water was established in the host lab which will be used in subsequent studies. We examined metabolic responses such as expended energy, preferred usage of nutrients, glucose tolerance - referred to as metabolic phenotyping - and mitochondrial function of different tissues. We could show that NAD supplementation induced slight effects on energy metabolism, that were different in the two mouse models. Mitochondrial oxygen usage of cardiac muscle fibres as a measure for mitochondrial function was increased upon NAD supplementation in one, but not the other mouse strain. Using NAD metabolomics as a method which is currently being established in the host lab for quantitating the exact amounts of NAD and NAD intermediates in different tissues, we will find out how much of the supplemented NAD precursor has arrived in what tissue. This is part of an ongoing collaboration between the fellow and host.

Final results

The study of the effects of the chemotherapeutic agent on mitochondrial function in a cellular model of hepatocarcinoma is highly relevant for elucidating the resistance mechanism to this chemotherapeutic, which is frequently occurring in patients with hepatocarcinoma.

The direct comparison of the responses of the mouse strains to NAD supplementation is to our knowledge the first study of this type being performed. This knowledge is important both for the correct choice of mouse strains for future research in the field of energy metabolism and NAD supplementation, but also a basis to elucidate the way that NAD supplementation works both in mice and humans. Since NAD supplements are being freely available and are advertised as anti-aging and anti-obesity factors, greater knowledge about the mechanism of action of these supplements is urgently needed and is of growing interest also for the general public.

During the 18 months fellowship the work has been well received by the scientific community.
Results from part 1 were presented at the meeting â€œMitochondria: form and function.â€ in London in Sept. 2017 and at the German Society for Endocrinology meeting in March 2018. Publication is planned for summer 2018. Results from the second part of the project were presented at an NAD workshop in Copenhagen, Denmark in Nov. 2017 and at the German Society for Endocrinology meeting in March 2018. Publication is also planned for summer 2018.

The Fellow now has achieved a senior role at the Collaborative Research Centre (SFB1052) Leipzig and is focusing on understanding the underlying mechanisms of ectopic fat accumulation. Dr. Garten has also started working towards her next academic degree (Dr. Sci.) to become eligible for teaching at German Universities.