University of Pittsburgh Division of Endocrinology and Metabolism R01 Study Examines How Intestinal CFTR Dysfunction Disrupts Dietary Fat Absorption in Cystic Fibrosis

Alison B. Kohan, PhD, FAHA, associate professor of medicine and immunology in the Division of Endocrinology and Metabolism at the University of Pittsburgh School of Medicine, is leading a National Institutes of Health-funded R01 research project to investigate why people with cystic fibrosis can continue to experience fat malabsorption despite pancreatic enzyme replacement therapy.

Dr. Kohan and her co-PI Craig A. Hodges, PhD, of Case Western Reserve University, project is titled “Deciphering the Role of the Cystic Fibrosis Transmembrane Regulator (CFTR) in Dietary Fatty Acid Transport in the Small Intestine,” is examining how CFTR dysfunction impairs dietary fatty acid transport across the small intestinal epithelium.

The studies will also evaluate whether elexacaftor-tezacaftor-ivacaftor (ETI; Trikafta) therapy can correct the classic fat malabsorption defect that is frequently observed in patients with CF and Drs. Kohan and Hodges will determine if changing the type of fat in the diet may improve this phenotype. Dr. Kohan and colleagues will combine mechanistic studies in animal models of CF (in mice expressing the human CFTR mutations), novel surgical techniques that allow for the direct collection of intestinal lymph and the dietary fat that is found there, and experiments using primary intestinal organoids derived from human tissue. Together, these mouse and human molecular approaches will help Drs. Kohan and Hodges determine the nature of the CF intestinal defect and potential dietary approaches to resolving it. Their proposal will also help to address an open question in the field, which is whether ETI therapy will be as successful at reducing CF symptoms in the gut as it has been at reducing symptoms in the lung.

“ETI has radically changed pulmonary care for many people living with CF, but we still need to understand how it affects the gut and why the intestinal response may differ among patients,” Dr. Kohan says.

A Persistent Nutritional Problem in Cystic Fibrosis

Although cystic fibrosis (CF) is most commonly associated with pulmonary disease and lung complications, the earliest signs of CF in newborns and young children occur in the gastrointestinal system. CFTR dysfunction affects the intestine, pancreas, and biliary system and can lead to intestinal obstruction, pancreatic insufficiency, dietary fat malabsorption, poor growth, and low body weight long before lung complications begin to manifest.

“Pancreatic enzyme therapy has changed the clinical course of CF by allowing children to survive the severe nutritional complications that had once caused many to die in infancy. However, it does fully correct malnutrition, fat malabsorption, or poor growth. We’ve learned that these intestinal symptoms, while rarely life-threatening, are closely tied to how severe a patient’s lung disease becomes and their overall lifespan,” Dr. Kohan says.

Pancreatic enzyme replacement therapy addresses one important source of malabsorption by giving patients with CF back the enzymes that their pancreas cannot adequately secrete (due to fibrosis of the pancreatic ducts). These enzymes are necessary to break dietary triglycerides into free fatty acids and simpler lipid products that can be absorbed by the intestine. Traditionally, people with CF have also been told to consume a high-fat diet to compensate for incomplete absorption of these necessary nutrients — essentially flooding the system with as much fat as possible to increase the chances that more will be absorbed by the body.

“The problem is that many people with CF continue to have nutritional and growth deficits despite pancreatic enzymes and a high-fat diet. This means that enzyme replacement isn’t enough, and we have been missing an important additional mechanism behind these defects,” Dr. Kohan says.

Therefore, pancreatic insufficiency is likely only one part of the problem. Even after dietary triglycerides have been digested by pancreatic enzymes, the resulting fatty acids have to move across the cell membrane of enterocytes, the key fat absorptive cell in the intestine, and then that dietary fat is reassembled and secreted across another cell membrane packaged within chylomicron particles. These chylomicrons are key transport vehicles that carry all of our dietary fat from the intestine and deliver it to other cells and tissues so the fat can be used for important metabolic processes. A defect anywhere along this pathway (across multiple membranes and through packaging and secretion) could limit the amount of dietary fat available to the body, leading to systemic growth and nutrition problems.

Identifying a Defect Beyond Pancreatic Insufficiency

Dr. Kohan and colleagues’ R01 study builds on a 2024 study the lab published in the Journal of Lipid Research. In this research, Dr. Kohan’s lab used CF mouse models to look at dietary fat absorption, chylomicron production, and their downstream metabolism.

Dr. Kohan’s team initially expected to find undigested triglycerides accumulating in the intestinal lumen because of insufficient pancreatic enzymes. However, what they found instead was an accumulation of free fatty acids and other triglyceride breakdown products. The dietary fat was digested but was not moving normally across the intestinal epithelium and into the enterocytes.

“Once we saw that the dietary fat breakdown products were not crossing the cell membrane into the enterocytes, we began thinking about how CFTR dysfunction might be interfering with fat transport beyond traditional mechanisms. The role of an ion transporter had been something we had totally ignored – but it seemed to have a major impact of the movement of lipids, ” Dr. Kohan says.

Without sufficient fatty acids inside the enterocytes, the cells lack the ability to produce normal chylomicrons. The CF animal models secreted smaller particles containing less triglyceride, and those abnormal chylomicrons were cleared from plasma into extraintestinal tissues approximately 10 times faster than chylomicrons produced by models without CF. This pattern differs dramatically from healthy chylomicrons, which stay in the circulation for a longer time and can deliver their dietary fat cargo to growing and active tissues.

These results suggested a connected series of problems. Dysregulated fatty acid transport across the intestinal epithelium reduced the amount of dietary fat available inside the enterocytes, which disrupted normal chylomicron formation, and changed their metabolism.

Testing How CFTR Dysfunction Interferes with Fatty Acid Transport

Dr. Kohan and colleagues’ current R01 study is designed to determine why digested fatty acids remain within the intestinal lumen rather than moving efficiently into enterocytes.

“We are examining whether changes in intestinal pH or mucus, both of which are changed by impaired CFTR ion transport, prevent these lipid products from crossing into the enterocytes,” Dr. Kohan says.

The research also will test how different types of dietary fat which are absorbed through a chylomicron-independent mechanism could be a potential workaround to this defect. If intestinal epithelial uptake is already limited, increasing the amount of dietary fat that moves through a different pathway could supply additional fat to the enterocytes and ultimately lipid-requiring tissues.

The project will also try to determine if ETI therapy can improve fatty acid transport, chylomicron formation, or both. This is a question that CF clinicians and scientists are excited to determine, since the epidemiologic studies on this topic have had very different findings on this topic.

Measuring Chylomicrons at the Point of Secretion

A specialized surgical intestinal lymph-collection model used in Dr. Kohan’s laboratory allows the team to measure chylomicrons immediately after they leave the intestine.

Chylomicrons do not pass directly from enterocytes into the bloodstream. They are first secreted into the intestinal lymphatic system, which later empties into the systemic circulation near the subclavian vein. Once chylomicrons enter the blood, enzymes and tissues begin removing their lipid cargo and using them as needed for various biological processes and cellular needs.

“By collecting intestinal lymph, we can measure chylomicrons at the time they are secreted and directly examine aspects of their synthesis, secretion, and key characteristics. Lymph is something we can’t study directly in people, so the specialized collection process we have developed for our animal models is very valuable, Dr. Kohan says.

The method allows Dr. Kohan and colleagues to determine how rapidly chylomicrons are produced, how much triglyceride they contain, how large they are, and how they subsequently behave in the circulation.

“Human lymph samples are rare, and the ones that do exist are small in volume and generally collected at a single time point. They do not provide enough material to answer the kinds of mechanistic questions we are asking along multiple time points during dietary fat absorption,” Dr. Kohan says.

Connecting the Experimental Findings With Human Intestinal Biology

To determine whether the epithelial defect identified in the animal models also is present in human tissue, Dr. Kohan and colleagues will use primary intestinal organoids derived from individuals with and without cystic fibrosis.

“We can grow primary intestinal organoids from healthy and CF tissue and use them to directly test the movement of fat across the intestinal membrane,” Dr. Kohan says.

The animal and organoid models serve complementary purposes. Animal studies allow controlled dietary interventions, tracer experiments, serial intestinal lymph collection, and evaluation of chylomicron metabolism in an intact physiological system. Human organoids let the team test epithelial transport mechanisms in human cells.

“We think this combination of biochemical studies, clinically relevant diets, and work in human intestinal cells will help us understand what is happening in the CF intestine and may allow us to propose different kinds of dietary approaches for further study,” Dr. Kohan says.

References and Further Reading

• The Kohan Lab
• Deciphering the Role of the Cystic Fibrosis Transmembrane Regulator (CFTR) in Dietary Fatty Acid Transport in the Small Intestine. NIH Project Number: R01DK143623.
• Teng L, et al. Impaired intestinal free fatty acid transport followed by chylomicron malformation, not pancreatic insufficiency, cause metabolic defects in cystic fibrosis. J Lipid Res. 2024; 65(7): 100551.
• Dedousis NL, Teng L, Kohan AB. The Isolation of Flowing Mesenteric Lymph in Mice to Quantify In Vivo Kinetics of Dietary Lipid Absorption and Chylomicron Secretion. J Vis Exp. 2022; Nov 30; (189).
• Dedousis N, Teng L, Kanshana JS, Kohan AB. A single-day mouse mesenteric lymph surgery in mice: an updated approach to study dietary lipid absorption, chylomicron secretion, and lymphocyte dynamics. J Lipid Res. 2022; 63(11): 100284.
• Li D, Rodia CN, Johnson ZK, Bae M, Muter A, Heussinger AE, Tambini N, Longo AM, Dong H, Lee JY, Kohan AB. Intestinal basolateral lipid substrate transport is linked to chylomicron secretion and is regulated by apoC-III. J Lipid Res. 2019; 60(9): 1503-1515.