Cholesterol is not the only lipid involved in trans fat-driven cardiovascular disease

Excess cholesterol is known to form artery-clogging plaques that can lead to stroke, arterial disease, heart attack, and more, making it the focus of many heart health campaigns. Fortunately, this attention to cholesterol has prompted the development of cholesterol-lowering drugs called statins and lifestyle interventions like dietary and exercise regimens. But what if there's more to the picture than just cholesterol?

New research from Salk Institute scientists describes how another class of lipids, called sphingolipids, contributes to arterial plaques and atherosclerotic cardiovascular disease (ASCVD). Using a longitudinal study of mice fed high-fat diets -- with no additional cholesterol -- the team tracked how these fats flow through the body and found the progression of ASCVD induced by high trans fats was fueled by the incorporation of trans fats into ceramides and other sphingolipids. Knowing that sphingolipids promote atherosclerotic plaque formation reveals another side of cardiovascular disease in addition to cholesterol.

The findings, published in Cell Metabolism on November 14, 2024, open an entirely new avenue of potential drug targets to address these diseases and adverse health events like stroke or heart attacks.

"Fat is a major component of our diet, and eating trans fats is known to drive heart disease. We used this phenomenon to understand the biological mechanisms putting us at risk," says senior author Christian Metallo, professor and holder of the Daniel and Martina Lewis Chair at Salk. "There have been lots of studies investigating how trans fats drive cardiovascular risk, but it always comes back to cholesterol -- we wanted to take another look that omits cholesterol as a factor, and we found an enzyme and pathway relevant to cardiovascular disease that we can potentially target therapeutically."

When dietary fats enter the body through the foods we eat, they must be sorted and processed into compounds called lipids, such as triglycerides, phospholipids, cholesterol, or sphingolipids. Lipoproteins -- like the familiar HDL, LDL, and VLDL -- are used to transport these lipids through the blood.

Sphingolipids have become useful biomarkers for diseases like ASCVD, non-alcoholic fatty liver disease, obesity, diabetes, peripheral neuropathy, and neurodegeneration. However, it is unclear exactly how the incorporation of different dietary fats into sphingolipids leads to the development of ASCVD.

In particular, the researchers were curious to ask how the processing of trans fats into sphingolipids may be creating atherosclerotic plaques. They wondered, could sphingolipids created in the liver influence the secretion of lipoproteins like VLDL into the bloodstream that, in excess, cause arterial blockages?

The fate of dietary fat is often determined by the protein that metabolizes it, explains Metallo, so it was important for the Salk team to first explore the metabolic landscape that creates sphingolipids in the first place. They started their survey with a protein called SPT, which acts as a floodgate to regulate the synthesis of sphingolipids from fat molecules and amino acids (other cellular building blocks) like serine.

The team suspected that trans fats were being incorporated into sphingolipids by SPT, which, in turn, would promote the excess lipoprotein secretion into the bloodstream that causes ASCVD.

To test their theory, they compared the processing of two different fats, cis fats and trans fats. The difference between these two comes down to the placement of a hydrogen atom; cis fats, found in natural foods like fish or walnuts, have a kink in their structure caused by two side-by-side hydrogen atoms, whereas trans fats, found in processed foods like margarine or anything fried, have a straight-chain structure caused by two opposing hydrogen atoms. Importantly, the kink in cis fats means they cannot be tightly packed -- a positive feature for avoiding impenetrable clogs.

The researchers combined mouse model dietary manipulation with metabolic tracing, pharmacological interventions, and physiological analyses to answer their question -- what is the link between trans fats, sphingolipids, and ASCVD?

"We found the incorporation of trans fats through SPT increased lipoprotein secretion from the liver, which then promoted the formation of atherosclerotic plaques," says first author Jivani Gengatharan, a postdoctoral researcher in Metallo's lab. "This highlights sphingolipid metabolism as a key node in the progression of cardiovascular disease driven by specific dietary fats."

Starting with cells in Petri dishes, the team looked at whether trans or cis fats were preferentially metabolized by SPT -- and it turns out that SPT preferred trans fats. Furthermore, SPT's bias for trans fats was causing downstream sphingolipid secretion that could go on to cause plaque formation.

Then, they moved from Petri dishes to mice, and Gengatharan designed otherwise identical diets containing either high trans or high cis fats but little cholesterol, feeding them to mice for 16 weeks. In the end, they saw mice consuming a high trans fat diet were producing trans fat-derived sphingolipids that promoted the secretion of VLDL from the liver into the bloodstream. This, in turn, accelerated the buildup of atherosclerotic plaques and the development of fatty livers and insulin dysregulation. High cis fat diet mice, on the other hand, experienced shorter-term, less harmful effects like weight gain.

To probe these effects further, they inhibited SPT to see whether they could limit negative trans fat effects in mice, finding that reducing SPT activity did decrease trans fat-induced atherosclerosis. According to Metallo, these findings make this sphingolipid synthesis pathway through SPT a critical target for ASCVD therapeutics moving forward.

"As we get a better grasp on identifying and measuring these diverse circulating molecules in our bodies and how they're metabolized, we could make huge strides in personalizing medicine accordingly," says Metallo. "For now, I recommend everything in moderation -- we all have our own diets and genetics and predispositions. As we explore and understand those factors, we can improve our knowledge and expand treatment options in the future."

One particular SPT subunit stood out to the researchers as the subject of future research, since the team suspects it is responsible for selectively spitting dangerous lipids out of the liver. With the spotlight on SPT, the team hopes to see new non-statin drug development plans for managing and preventing cardiovascular disease.

Despite the World Health Organization (WHO) announcing a plan to eliminate trans fats from food supplies by the end of 2023, nearly 4 billion people remain at risk in 2024 due to countries not abiding by WHO's best practices. The team hopes their work can make a difference in the lives of individuals still at risk.

Other authors include Zoya Chih, Maureen Ruchhoeft, and Ethan Ashley of Salk; Michal Handzlik and Courtney Green of Salk and UC San Diego; Patrick Secrest and Philip Gordts of UC San Diego; and Martina Wallace of University College Dublin.

The work was supported by the National Institutes of Health (R01CA234245), Aileen S. Andrew Foundation, and Mary K. Chapman Foundation.