Okayama University research: Lack of enzyme promotes fatty liver disease in thin patients‏

 Researchers observe protection against obesity and insulin resistance but at the cost of prominent fatty liver disease in mice lacking the PEMT enzyme and patients with low levels of PEMT.

Non-alcoholic fatty liver disease – steatohepatitis - is an increasingly common chronic form of hepatitis. As Jun Wada and colleagues at Okayama University Graduate School of Medicine, Shigei Medical Research Institute and Dainippon Sumitomo Pharma explain in a recent report, “Although obesity is undoubtedly one of the main risk factors for the development of non-alcoholic fatty liver disease, many clinical observations demonstrated the presence of lean NAFLD patients with normal body mass index (BMI).” The team’s latest work shows that absence of the enzyme phosphatidylethanolamine N-methyltransferase (PEMT), while protecting from diet-induced obesity and diabetes, leads to the prominent development of fatty liver disease and tumours in response to a high-fat high-sucrose diet.

PEMT catalyses methylation of phosphatidylethanolamine (PE) to phosphatidylcholine (PC) in liver cells using S-adenosyl methionine as a methyl donor. The ratio of PE to PC is known to be crucial to cell membrane integrity and resistance to endoplasmic reticulum stress and the infiltration of the liver with fat. 

Wada and colleagues fed mice lacking the PEMT enzyme a high-fat high-sucrose diet for up to 90 weeks. They monitored the fat accumulation and insulin resistance before dissecting them to examine their livers. The researchers noted enhanced apoptosis and cell proliferation in the liver, which they explain through transactivation of the protein p53, which is inhibited in the presence of PEMT. The researchers also note a similarity in the response of the PEMT knock-out mice to that of lean human patients suffering from non-alcoholic steatohepatitis.

The researchers then performed genome-wide sequencing-based DNA methylation analysis. Further investigation revealed that PEMT mRNA expression in liver tissues of human non-alcoholic steatohepatitis patients was significantly lower than for simple steatosis, and as they add “lower quartiles of PEMT mRNA demonstrated lower BMI and platelet counts, suggesting lower expression of PEMT is critically linked to the pathogenesis of lean non-alcoholic steatohepatitis.”

Background
Non-alcoholic fatty liver disease (NAFLD)
Extensive build-up of fat in the liver is responsible for a range of diseases including simple steatosis and non-alcoholic steatohepatitis (NASH). The increased instances of NAFLD are linked with the rise in obesity and diabetes, which are now considered to have reached epidemic proportions. However, NASH has also been reported in patients with a normal body mass index (BMI), and the prevalence of “lean NAFLD” was recently reported at 12% in Greece, 20% in India and 15% in China.

Choline deficiency and lean NAFLD
Mice fed a methionine- and choline-deficient diet (MCD) are a widely used mouse models for NASH research. An absence of obesity and insulin resistance has been observed in both MCD mice and PEMT knockout mice fed high-fat high-sucrose diets.

PC is synthesized from choline, and a balance in PE and PC levels is thought to be important for maintaining cell membrane integrity, stabilising lipid droplets and the normal distribution of fat. Disrupting this balance appears to cause accumulation of fat in the liver. 

PEMT catalyses methylation of PE, which also produces PC. The similar effects – lack of obesity and prominent steatohepatitis – is also observed in PEMT knock-out mice and human NAFLD patients with low levels of PEMT.

DNA methylation analysis
The genome-wide sequencing-based DNA methylation analysis by the researchers revealed enhanced methylation of two genes associated with cyclin D1 degradation and negative regulation. These results implicate the upregulation of cyclin D1 in the development of liver disease and tumours in PEMT knock-out mice.

 Caption

Phenotype of Pemt+/+, Pemt+/− and Pemt−/− mice under high fat-high sucrose (HFHS)
diet at 60 weeks of age. (a–c) Gross appearance of liver. Bar = 1 cm. Regenerative nodules and adenoma are indicated by arrow heads (c). (d–f) Masson-Trichrome staining of liver tissues. Bar = 100 μ m.

Reference 
Atsuko Nakatsuka, Makoto Matsuyama, Satoshi Yamaguchi, Akihiro Katayama, Jun Eguch, Kazutoshi Murakami, Sanae Teshigawara, Daisuke Ogawa, Nozomu Wada, Tetsuya Yasunaka, Fusao Ikeda, Akinobu Takaki, Eijiro Watanabe & Jun Wada. Insufficiency of phosphatidylethanolamine N-methyltransferase is risk for lean non-alcoholic steatohepatitis, Scientific Reports 6 21721 (2016). 
DOI: 10.1038/srep21721
http://www.nature.com/articles/srep21721

Enzyme potential target for fight against obesity and diabetes

Removing an enzyme that controls bile acid and hormone levels significantly protects female mice from weight gain, according to a new study presented today at the Society for Endocrinology annual conference in Edinburgh. The finding offers a new a therapeutic target in the fight against obesity.

Steroid hormones and bile acids have multiple functions that affect appetite, physical activity and how energy is used and stored in the body. For example, the sex hormone oestrogen (a steroid) has previously shown to decrease women’s appetite while firing up their metabolism and levels of physical activity. Bile acids are important to digest fats in diets, without which animals could not make the most out of a fatty food’s calorific content.

The enzyme 5β-Reductase helps generate bile acid and clears excess levels of steroid hormones in the human body.

In this study, researchers from the University of Oxford compared the effects of feeding wild mice a high calorie, fat-rich diet with mice that lacked the ability to make 5β-Reductase over a period of 30 weeks.

Female mice without 5β-Reductase gained 42% less weight than the wild mice (15.8g vs 27.2g respectively), while males in both experimental groups gained the same amount of weight. Female mice without 5β-Reductase also stored less fat around the gonads, vital organs and under their skin compared to wild mice, while also being more sensitive to insulin and better at controlling their blood glucose levels.

“The gender-specific health outcomes of our experiment are interesting but poorly understood”, said lead author of the study Dr Laura Gathercole. “It could be that lacking this key enzyme means female mice are less able to extract energy from their food, spend more energy to power their metabolism, or both at the same time”.

“Tweaking steroid and bile acid levels has significant health implications and so 5β-Reductase could be an important potential therapeutic target in metabolic disease”, she continued.

The researchers next steps are to pinpoint the mechanisms behind the phenomenon, which could provide insights into the different ways males and females regulate their energy and metabolisms.

New Immune Defense Enzyme Discovered

A previously unknown serine protease forms part of the antibacterial defence arsenal of neutrophil granulocytes.

Neutrophil granulocytes comprise important defences for the immune system. When pathogenic bacteria penetrate the body, they are the first on the scene to mobilise other immune cells via signal molecules, thereby containing the risk. To this end, they release serine proteases – enzymes that cut up other proteins to activate signal molecules. Scientists at the Max Planck Institute of Neurobiology in Martinsried have now discovered a new serine protease: neutrophil serine protease 4, or NSP4. This enzyme could provide a new target for the treatment of diseases that involve an overactive immune system, such as rheumatoid arthritis.
The functioning of the immune system is based on the complex interplay of the most diverse cells and mediators. For example, neutrophil granulocytes (a group of specialized white blood cells) react to bacteria by releasing substances called serine proteases. These enzymes are able to activate signal molecules, such as the chemokines, by cleaving them at a specific position on the molecule. The active signal molecules then guide other immune cells to the focus of inflammation in order to destroy the pathogens.
A research team led by Dieter Jenne at the Max Planck Institute of Neurobiology in Martinsried has come across a previously unknown protease in humans: neutrophil serine protease 4, or NSP4. "The special thing about this enzyme is that it cuts proteins that have the amino acid arginine at a particular point", says Dieter Jenne, research group leader at the Martinsried-based Institute. "This is where NSP4 differs from the other three known neutrophil serine proteases, which are similar in molecular structure, but have a different recognition motif." The scientists may be able to harness this difference to develop an active substance that specifically inhibits NSP4, thereby reducing the immune reaction.
However, serine protease activity comes at a cost. The enzymes not only heal inflammations, but sometimes cause them in the first place. If too many immune cells are activated, they can use their arsenal of aggressive chemical weapons against the body's own tissues. A number of chronic inflammatory diseases are based on precisely this effect. As a result, scientists are searching for substances that can block the neutrophil proteases. To date, however, none of the substances tested have been developed into effective drugs.
"So far, we don't know the identity of the NSP4 substrate, but we assume they must be signal molecules", says Dieter Jenne. Activated chemokines can recruit a vast number of neutrophils, and their sheer quantity alone is enough to cause tissue damage. "Proteases sometimes act as accelerants and can even trigger a chronic inflammation quite independently of bacterial intruders. If we dampened down the defences, we could counteract this effect", explains the scientist.
In terms of evolutionary history, NSP4 is the oldest of the four known neutrophil serine proteases. Using gene sequences, scientists have shown that the enzyme has hardly changed through hundreds of millions of years of evolution from bony fish to humans. "That would indicate that NSP4 regulates a fundamental process", says Dieter Jenne.
The fact that the enzyme remained undiscovered until now is because it occurs at a much lower concentration than the other three proteases. The Max Planck scientists came across it while searching the human genome for genes that encode serine proteases. In the process, they noticed a previously unknown gene sequence. Natascha C. Perera, a member of the Martinsried research group and lead author of the study, managed to produce and examine the enzyme in its active, folded state.
If they are to establish NSP4 in the future as a possible target protein for anti-inflammatory drugs, the scientists must now examine its function in living organisms and discover whether blocking the enzyme has adverse effects. The scientists are working with the company Novartis to answer these questions in laboratory mice. "NSP4 inhibitors could be used in diseases like chronic arthritis or inflammatory skin diseases", says Dieter Jenne, "but first we have to test the long-term effects of these substances."

**Published in "SCIENCE DAILY"

enlarge

Microscope image of normal human bone marrow tissue with stained NSP4 in myeloblasts and myelocytes. (Credit: MPI of Neurobiology)