Anti-Inflammatory and Therapeutic Effects of a Novel Small-Molecule Inhibitor of Inflammation in a Male C57BL/6J Mouse Model of Obesity-Induced NAFLD/MAFLD.

Purpose: Non-alcoholic fatty liver disease (NAFLD), recently renamed metabolic (dysfunction) associated fatty liver disease (MAFLD), is the most common chronic liver disease in the United States. Presently, there is an intense and ongoing effort to identify and develop novel therapeutics for this disease. In this study, we explored the anti-inflammatory activity of a new compound, termed IOI-214, and its therapeutic potential to ameliorate NAFLD/MAFLD in male C57BL/6J mice fed a high fat (HF) diet. Methods: Murine macrophages and hepatocytes in culture were treated with lipopolysaccharide (LPS) ± IOI-214 or DMSO (vehicle), and RT-qPCR analyses of inflammatory cytokine gene expression were used to assess IOI-214's anti-inflammatory properties in vitro. Male C57BL/6J mice were also placed on a HF diet and treated once daily with IOI-214 or DMSO for 16 weeks. Tissues were collected and analyzed to determine the effects of IOI-214 on HF diet-induced NAFL D/MAFLD. Measurements such as weight, blood glucose, serum cholesterol, liver/serum triglyceride, insulin, and glucose tolerance tests, ELISAs, metabolomics, Western blots, histology, gut microbiome, and serum LPS binding protein analyses were conducted. Results: IOI-214 inhibited LPS-induced inflammation in macrophages and hepatocytes in culture and abrogated HF diet-induced mesenteric fat accumulation, hepatic inflammation and steatosis/hepatocellular ballooning, as well as fasting hyperglycemia without affecting insulin resistance or fasting insulin, cholesterol or TG levels despite overall obesity in vivo in male C57BL/6J mice. IOI-214 also decreased systemic inflammation in vivo and improved gut microbiota dysbiosis and leaky gut. Conclusion: Combined, these data indicate that IOI-214 works at multiple levels in parallel to inhibit the inflammation that drives HF diet-induced NAFLD/MAFLD, suggesting that it may have therapeutic potential for NAFLD/MAFLD.

Metabolic profiling reveals dysregulated lipid metabolism and potential biomarkers associated with the development and progression of Fragile X-Associated Tremor/Ataxia Syndrome (FXTAS).

Fragile X-associated Tremor/Ataxia Syndrome (FXTAS) is a neurodegenerative disorder associated with the FMR1 premutation. It is currently unknown when, and if, individual premutation carriers will develop FXTAS. Thus, with the aim of identifying biomarkers for early diagnosis, development, and progression of FXTAS, we performed global metabolomic profiling of premutation carriers (PM) who, as part of an ongoing longitudinal study, emerged into two distinct categories: those who developed symptoms of FXTAS (converters, CON) at subsequent visits and those who did not (non-converters, NCON) and we compared to age-matched healthy controls (HC). We assessed CGG repeat allele size by Southern Blot and PCR analysis. Metabolomic profile was obtained by ultra-performance liquid chromatography, accurate mass spectrometer, and an Orbitrap mass analyzer. In this study we found 47 metabolites were significantly dysregulated between HC and the premutation groups (PM). Importantly, we identified 24 metabolites that showed significant changes in expression in the CON as compared to the NCON both at V1 and V2, and 70 metabolites in CON as compared to NCON but only at V2. These findings suggest the potential role of the identified metabolites as biomarkers for early diagnosis and for FXTAS disease progression, respectively. Interestingly, the majority of the identified metabolites were lipids, followed by amino acids. To our knowledge, this the first report of longitudinal metabolic profiling and identification of unique biomarkers of FXTAS. The lipid metabolism and specifically the sub pathways involved in mitochondrial bioenergetics, as observed in other neurodegenerative disorders, are significantly altered in FXTAS.

Transcription factor ISX mediates the cross talk between diet and immunity.

The intestinal epithelium is a major site for the conversion of dietary ß-carotene to retinaldehyde by the enzyme BCO1. The majority of retinaldehyde is further metabolized to retinol (vitamin A), esterified and packaged into triacylglycerol-rich chylomicrons for bodily distribution. Some serve on-site for the synthesis of retinoic acid, a hormone-like compound, which exerts pleiotropic and dominant effects on gastrointestinal immunity. We report here that the intestine-specific homeobox protein ISX is critical to control the metabolic flow of ß-carotene through this important branching point of vitamin A metabolism. This transcription factor represses Bco1 gene expression in response to retinoic acid signaling. In ISX-deficient mice, uncontrolled Bco1 gene expression led to increased retinoid production in the intestine. Systemically, this production resulted in highly elevated hepatic retinoid stores. In the intestine, it increased the expression of retinoic acid-inducible target genes such as Aldh1a2, Dhrs3, and Ccr9 The ß-carotene-inducible disruption of retinoid homeostasis affected gut-homing and differentiation of lymphocytes and displayed morphologically in large lymphoid follicles along the intestine. Furthermore, it was associated with an infiltration of the pancreas by gut-derived lymphocytes that manifested as a pancreatic insulitis with ß-islet cell destruction and systemic glucose intolerance. Thus, our study identifies an important molecular interlink between diet and immunity and indicates that vitamin A homeostasis must be tightly controlled by ISX to maintain immunity and tolerance at the intestinal barrier.

Genetic dissection in a mouse model reveals interactions between carotenoids and lipid metabolism.

Carotenoids affect a rich variety of physiological functions in nature and are beneficial for human health. However, knowledge about their biological action and the consequences of their dietary accumulation in mammals is limited. Progress in this research field is limited by the expeditious metabolism of carotenoids in rodents and the confounding production of apocarotenoid signaling molecules. Herein, we established a mouse model lacking the enzymes responsible for carotenoid catabolism and apocarotenoid production, fed on either a ß-carotene- or a zeaxanthin-enriched diet. Applying a genome wide microarray analysis, we assessed the effects of the parent carotenoids on the liver transcriptome. Our analysis documented changes in pathways for liver lipid metabolism and mitochondrial respiration. We biochemically defined these effects, and observed that ß-carotene accumulation resulted in an elevation of liver triglycerides and liver cholesterol, while zeaxanthin accumulation increased serum cholesterol levels. We further show that carotenoids were predominantly transported within HDL particles in the serum of mice. Finally, we provide evidence that carotenoid accumulation influenced whole-body respiration and energy expenditure. Thus, we observed that accumulation of parent carotenoids interacts with lipid metabolism and that structurally related carotenoids display distinct biological functions in mammals.

Transport of vitamin A across blood-tissue barriers is facilitated by STRA6.

Vitamin A bound to retinol binding protein 4 (RBP4) constitutes the major transport mode for retinoids in fasting circulation. Emerging evidence suggests that membrane protein, STRA6 (stimulated by retinoic acid 6), is the RBP4 receptor and vitamin A channel; however, the role of STRA6 in vitamin A homeostasis remains to be defined in vivo We subjected Stra6-knockout mice to diets sufficient and insufficient for vitamin A and used heterozygous siblings as controls. We determined vitamin A levels of the eyes, brain, and testis, which highly express Stra6, as well as of tissues with low expression, such as lung and fat. We also studied the consequence of STRA6 deficiency on retinoid-dependent processes in tissues. Furthermore, we examined how STRA6 deficiency affected retinoid homeostasis of the aging mouse. The picture that emerged indicates a critical role for STRA6 in the transport of vitamin A across blood-tissue barriers in the eyes, brain, and testis. Concurrently, fat and lung rely on dietary vitamin A. In testis and brain, Stra6 expression was regulated by vitamin A. In controls, this regulation reduced vitamin A consumption when the dietary supply was limited, sequestering it for the eye. Thus, STRA6 is critical for vitamin A homeostasis and the adaption of this process to the fluctuating supply of the vitamin.-Kelly, M., Widjaja-Adhi, M. A. K., Palczewski, G., von Lintig, J. Transport of vitamin A across blood-tissue barriers is facilitated by STRA6.

Characterization of the Role of ß-Carotene 9,10-Dioxygenase in Macular Pigment Metabolism.

A family of enzymes collectively referred to as carotenoid cleavage oxygenases is responsible for oxidative conversion of carotenoids into apocarotenoids, including retinoids (vitamin A and its derivatives). A member of this family, the ß-carotene 9,10-dioxygenase (BCO2), converts xanthophylls to rosafluene and ionones. Animals deficient in BCO2 highlight the critical role of the enzyme in carotenoid clearance as accumulation of these compounds occur in tissues. Inactivation of the enzyme by a four-amino acid-long insertion has recently been proposed to underlie xanthophyll concentration in the macula of the primate retina. Here, we focused on comparing the properties of primate and murine BCO2s. We demonstrate that the enzymes display a conserved structural fold and subcellular localization. Low temperature expression and detergent choice significantly affected binding and turnover rates of the recombinant enzymes with various xanthophyll substrates, including the unique macula pigment meso-zeaxanthin. Mice with genetically disrupted carotenoid cleavage oxygenases displayed adipose tissue rather than eye-specific accumulation of supplemented carotenoids. Studies in a human hepatic cell line revealed that BCO2 is expressed as an oxidative stress-induced gene. Our studies provide evidence that the enzymatic function of BCO2 is conserved in primates and link regulation of BCO2 gene expression with oxidative stress that can be caused by excessive carotenoid supplementation.

Evidence for compartmentalization of mammalian carotenoid metabolism.

The critical role of retinoids (vitamin A and its derivatives) for vision, reproduction, and survival has been well established. Vitamin A is produced from dietary carotenoids such as ß-carotene by centric cleavage via the enzyme BCO1. The biochemical and molecular identification of a second structurally related ß-carotene metabolizing enzyme, BCO2, has led to a prolonged debate about its relevance in vitamin A biology. While BCO1 cleaves provitamin A carotenoids, BCO2 is more promiscuous and also metabolizes nonprovitamin A carotenoids such as zeaxanthin into long-chain apo-carotenoids. Herein we demonstrate, in cell lines, that human BCO2 is associated with the inner mitochondrial membrane. Different human BCO2 isoforms possess cleavable N-terminal leader sequences critical for mitochondrial import. Subfractionation of murine hepatic mitochondria confirmed the localization of BCO2 to the inner mitochondrial membrane. Studies in BCO2-knockout mice revealed that zeaxanthin accumulates in the inner mitochondrial membrane; in contrast, ß-carotene is retained predominantly in the cytoplasm. Thus, we provide evidence for a compartmentalization of carotenoid metabolism that prevents competition between BCO1 and BCO2 for the provitamin and the production of noncanonical ß-carotene metabolites.

Mammalian carotenoid-oxygenases: key players for carotenoid function and homeostasis.

Humans depend on a dietary intake of lipids to maintain optimal health. Among various classes of dietary lipids, the physiological importance of carotenoids is still controversially discussed. On one hand, it is well established that carotenoids, such as ß,ß-carotene, are a major source for vitamin A that plays critical roles for vision and many aspects of cell physiology. On the other hand, large clinical trials have failed to show clear health benefits of carotenoids supplementation and even suggest adverse health effects in individuals at risk of disease. In recent years, key molecular players for carotenoid metabolism have been identified, including an evolutionarily well conserved family of carotenoid-oxygenases. Studies in knockout mouse models for these enzymes revealed that carotenoid metabolism is a highly regulated process and that this regulation already takes place at the level of intestinal absorption. These studies also provided evidence that ß,ß-carotene conversion can influence retinoid-dependent processes in the mouse embryo and in adult tissues. Moreover, these analyses provide an explanation for adverse health effects of carotenoids by showing that a pathological accumulation of these compounds can induce oxidative stress in mitochondria and cell signaling pathways related to disease. Advancing knowledge about carotenoid metabolism will contribute to a better understanding of the biochemical and physiological roles of these important micronutrients in health and disease. This article is part of a Special Issue entitled Retinoid and Lipid Metabolism.