Discovering carotenoid biodistribution pathways: Novel mechanisms and their implications for health

Miller, Anthony Paul

Permalink

https://hdl.handle.net/2142/121944 Copy

Description

Title

Discovering carotenoid biodistribution pathways: Novel mechanisms and their implications for health

Author(s)

Miller, Anthony Paul

Issue Date

2023-11-09

Director of Research (if dissertation) or Advisor (if thesis)

Amengual, Jaume

Doctoral Committee Chair(s)

Erdman, John

Committee Member(s)

• Chen, Hong
• von Lintig, Johannes

Department of Study

Food Science & Human Nutrition

Discipline

Food Science & Human Nutrition

Degree Granting Institution

University of Illinois at Urbana-Champaign

Degree Name

Ph.D.

Degree Level

Dissertation

Keyword(s)

• Carotenoids
• Biodistribution
• Retinoids
• Lipids

Abstract

Carotenoids constitute a diverse class of lipids comprising over 1,000 compounds, imparting vibrant yellow to red hues to a range of fruits and vegetables. Dietary intake of carotenoid-rich foods, along with elevated plasma carotenoid levels, has been associated with favorable health outcomes, including a reduced risk of cardiometabolic diseases and cancer. The biological effects of carotenoids are diverse, stemming from their intact molecular structures or as metabolites resulting from carotenoid cleavage. A notable example is lutein, which accumulates in its intact form within the human eye, safeguarding against damage caused by blue light and delaying the onset of age-related macular degeneration. On the other hand, the influence of the pro-vitamin A carotenoid β-carotene, at least on adipose tissue, is wholly dependent on its conversion to vitamin A. The demand for the development of animal models that accurately replicate carotenoid distribution and accumulation observed in humans has increased over the past two decades. While humans efficiently accumulate substantial quantities of carotenoids in plasma and tissues, the prevailing experimental models employed in biomedical research often fail to replicate this phenotype. This inadequacy extends to wild-type mice, the most utilized animal model in biomedical research. Dietary investigations conducted using wild-type mice reveal their inherent tendency to extensively cleave carotenoids, resulting in negligible carotenoid storage even after prolonged exposure to supra-physiological doses. Consequently, wild-type mice do not serve as suitable models for studying the biological impacts of carotenoids in their intact form, nor for exploring the underlying mechanisms governing their tissue distribution. Although alternative animal models such as ferrets or non-human primates exhibit carotenoid metabolism patterns comparable to humans, incorporating these models into mechanistic studies would be hampered by technical and ethical constraints. As a result, certain studies have resorted to cell culture and in vitro models to investigate these mechanisms, despite encountering technical complexities and inherent limitations. Our studies are linked by a common objective: to study novel factors affecting carotenoid metabolism and biodistribution. The research described fills essential knowledge gaps that will enable the design of effective nutritional intervention strategies in the future. In Chapter 2, we aimed to investigate the effects of the synthetic retinoid fenretinide on carotenoid metabolism and distribution in vivo. Fenretinide binds the retinol-binding protein 4 (RBP4) impairing vitamin A transport to tissues. However, fenretinide also inhibits vitamin A formation in in vitro models by blocking the β-carotene oxygenase 1 (BCO1), which cleaves β-carotene to vitamin A. In separate experiments, we used mice lacking BCO1 or BCO2, the second enzyme involved in carotenoid cleavage, to isolate the effect of fenretinide on carotenoid cleavage in mice fed β-carotene, as well as carotenoid and vitamin E absorption. Our results show that fenretinide reduces tissue vitamin A stores accompanied by an increase in plasma and tissue β-carotene. We also show that fenretinide regulates intestinal carotenoid and vitamin E uptake by activating vitamin A signaling in the gut, which could result in vitamin A and carotenoid deficiency in patients taking fenretinide. Chapter 3 examines different factors influencing carotenoid absorption and cleavage. For the first time, we administered the fungal carotenoid neurosporaxanthin to animals. Neurosporaxanthin possesses a unique chemical structure: 1) It is relatively polar, containing a carboxylic group, and thanks to its structure, it 2) could form vitamin A. Bioavailability studies showed that neurosporaxanthin exhibits a greater bioavailability in comparison to β-cryptoxanthin and β-carotene. We went on to assess the cleavage mechanism of neurosporaxanthin. We performed enzymatic assays with purified BCO1 and BCO2, together with feeding studies in mice to evaluate the potential of neurosporaxanthin as a vitamin A precursor. Our results showed that BCO1 cleaves neurosporaxanthin to form vitamin A, opening new avenues for the development of functional foods containing this compound. Scavenger receptors such as the scavenger receptor class B type 1 (SR-B1) and the cluster of differentiation 36 (CD36) are believed to have a crucial role in the absorption of carotenoids in the intestine and their uptake by peripheral tissues. While extensive evidence exists for the involvement of SR-B1 in this process, there is a knowledge gap in the role of CD36 in the uptake of carotenoids in vivo. Our findings in Chapter 4 reveal that depletion of CD36 in mice leads to elevated levels of β-carotene in both plasma and tissues, independent of food intake and body weight. Through experiments with FITC-dextran, we discovered that this increase is attributed to a more permeable gastrointestinal barrier in CD36-deficient animals. We also examined carotenoid levels in the plasma of subjects carrying the rs3211938 allele of the CD36 gene, which is associated with approximately 50% reduced CD36 protein levels. Our preliminary data show a trend of higher total plasma carotenoid levels in subjects with lower CD36 expression in comparison to control individuals. Overall, our findings in mice and humans demonstrate that CD36 deficiency leads to an impaired gut barrier, resulting in elevated carotenoid levels in tissues and plasma. The translocation of carotenoids from the liver to peripheral tissues is believed to occur through their mobilization within very low-density lipoprotein (VLDL) particles. In plasma, VLDL catabolism leads to the formation of low-density lipoproteins (LDL), which are largely cleared by the LDL receptor (LDLR) expressed in most tissues and the liver. LDLR is also expressed on the basolateral membrane of enterocytes, where it mediates a relatively uncharacterized process named transintestinal cholesterol excretion. The objective of Chapter 5 was to determine the role of LDLR in the uptake and excretion of carotenoids. We examined carotenoid fecal elimination in Bco1-/- and Bco1-/-Ldlr-/- mice fed a Standard diet containing β-carotene. HPLC results showed that fecal and intestinal β-carotene in Bco1-/- mice were 2.5-fold higher than those observed in Bco1-/-Ldlr-/- mice. On the contrary, plasma β-carotene levels were increased in Bco1-/-Ldlr-/- mice while adipose tissue β-carotene levels were decreased compared to Bco1-/- control mice. Overexpression of LDLR in the liver of Bco1-/- mice resulted in increased hepatic β-carotene and decreased peripheral tissue β-carotene levels. This data indicates that LDLR is a bona fide mediator of carotenoid homeostasis in mammals. Studies suggest that high-density lipoprotein (HDL) particles deliver carotenoids such as lutein to the eye. However, the main role of apolipoprotein A1 (apoA-I), the main component of HDL, and HDL is to promote cholesterol efflux from extrahepatic tissues by interacting with the membrane transporters ATP-binding cassettes ABCA1 and ABCG1. In Chapter 6, we conducted studies to investigate if HDL participates in the delivery and efflux of carotenoids from peripheral tissues. We utilized our Bco1-/- mice as a “backbone” to deplete and increase apoA1/HDL levels. Mice with depleted apoA-I/HDL exhibited similar hepatic β-carotene levels compared to Bco1-/- control mice, whereas mice with increased apoA-I/HDL levels showed approximately a 2-fold elevation in hepatic β-carotene levels. Administration of purified human apoA-I resulted in depletion of plasma and adipose tissue β-carotene pools, while simultaneously increasing hepatic β-carotene stores. In cell culture experiments, the presence of purified apoA-I facilitated the efflux of β-carotene from cultured white adipose tissue (WAT) explants. Taken together, this study suggests that HDL plays a dual role in carotenoid delivery and efflux. Our studies delved into various aspects of carotenoid metabolism and biodistribution, yielding valuable insights into the intricate mechanisms governing the absorption, cleavage, transport, and elimination of these compounds. These findings significantly contribute to our fundamental understanding of these processes, establishing a solid groundwork for future investigations in this field.

Graduation Semester

2023-12

Type of Resource

Thesis

Copyright and License Information

Copyright 2023 Anthony P. Miller

Owning Collections