MLK3-deficient mice immune to liver injury from SAD

Nonalcoholic fatty liver disease (NAFLD) is one of the leading causes of liver injury worldwide and the most common cause of chronic liver disease among children in western nations. It includes several disorders associated with excess lipid deposition in hepatocytes, including nonalcoholic steatohepatitis (NASH), the more severe form of the disease. About one-fourth of pediatric patients with NAFLD have NASH that may progress to cirrhosis.

NAFLD was first recognized in children in 1983, and its prevalence has increased with the obesity epidemic, rising from around 4 percent to 10 percent over the last two decades. Both obesity and fatty liver are linked to two fixtures of the standard American diet — saturated fat and high-density fructose, which has unique metabolic effects that increase the risk of NASH.

In addition to obesity, NASH is strongly associated with insulin resistance and elevated serum levels of saturated free fatty acids. An excess of toxic fatty acids in the circulation lead to hepatocyte death, infiltration of inflammatory cells into the liver and fibrosis.

Weight loss, physical exercise, and reduction of dietary intake of fats and high-fructose corn syrup are the first line treatments for NASH. Metabolic improvement starts to occur with as little as a 10 percent weight loss, and after one year, patients can lose up to 50 percent of fat in the liver, effectively reversing the condition. But lifestyle changes are difficult to initiate or maintain for some patients and families, who may not recognize their benefits.

"I have seen some families recognize that weight loss is not just about looks but also about preventing serious health problems, and they commit to a diet and exercise regimen. But often, there are barriers to adherence to diet and exercise, and people need a lot of motivation," says Samar H. Ibrahim, M.B., Ch.B., a pediatric gastroenterologist and hepatologist at Mayo Clinic's campus in Rochester, Minnesota. "We need to follow patients closely every three months, which gives them an opportunity to have their weight checked and gives us an opportunity to emphasize how important it is that they stick to their regimen."

The difficulty of sustaining lifestyle changes gives impetus to the search for pharmacological therapies targeting cellular and molecular mechanisms that promote liver injury in NASH.

Dr. Ibrahim is interested in mixed lineage kinases (MLKs), a family of serine/threonine protein kinases that includes MLK1, MLK2 and MLK3. Studies have shown that MLK3 mediates C-Jun N-terminal kinase (JNK) activation in the liver, which is pivotal to the development of NASH.

"When we treated cells in vitro with an MLK inhibitor, they became resistant to fatty acid-induced death, so that was something we wanted to explore further," Dr. Ibrahim says.

Murine model

Dr. Ibrahim and her colleagues recently conducted a mouse model study, comparing MLK3-deficient mice with wild-type mice controls. The mice were randomized to receive regular chow or a high-fat, high-carbohydrate (HFHC) diet and drinking water high in fructose and sucrose. After 16 weeks, JNK phosphorylation was significantly reduced in MLK3-deficient mice fed the HFHC diet compared with controls, independent of body weight.

The MLK3-deficient mice on the HFHC diet experienced a 1.3-fold increase in liver apoptotic cells whereas the wild-type controls had three times that number. The MLK3-deficient mice also had reduced serum ALT values compared with wild-type HFHC-fed mice. Taken together, these data suggest that the MLK-deficient mice were protected against HPHC diet-induced hepatic steatosis and liver injury, Dr. Ibrahim says.

Also shown in the study were the injurious effects of a high-fat, high-carbohydrate diet on the liver in general. Compared with mice fed a high-fat diet only, mice fed the HFHC diet had increased hepatic oxidative stress, macrophage aggregation in the liver, and fibrogenesis and collagen deposition, despite similar calorie intake and weight gain.

"Fructose consumption seemed necessary to move the process from simple steatosis to fibrogenesis, although MLK3-deficient mice did have relative attenuation of all the injurious features of NASH in this model," Dr. Ibrahim says.

Study results appeared in the March 2014 edition of Liver International.

Inhibitor trials

Based on this and other studies, Dr. Ibrahim believes the MLK3 signaling pathway is a potential target for drug therapy. Her ultimate goal is to conduct a human clinical trial of a new MLK inhibitor currently in development for treatment of neurodegenerative diseases. The drug has proved effective in vitro and in animal models.

"It's not nearly ready for use in humans, but we want to use it in animals to see if we can prevent development of nonalcoholic steatohepatitis," she says. "In our first attempts, we had some problems with solubility. We're also not sure how long to give it, so we have to optimize administration and duration. Still, I'm feeling optimistic. It's at least a five-year process, but we want to do it the right way."

For more information

Ibrahim SH, et al. Mixed lineage kinase 3 deficient mice are protected against the high fat high carbohydrate diet-induced steatohepatitis. Liver International. 2014;34:427.