Humanized UGT1 Mice, Regulation of UGT1A1, and the Role of the Intestinal Tract in Neonatal Hyperbilirubinemia and Breast Milk-Induced Jaundice

Abstract

Neonatal hyperbilirubinemia and the onset of bilirubin encephalopathy and kernicterus result in part from delayed expression of UDP-glucuronosyltransferase 1A1 (UGT1A1) and the ability to metabolize bilirubin. It is generally believed that acute neonatal forms of hyperbilirubinemia develop due to an inability of hepatic UGT1A1 to metabolize efficiently bilirubin for clearance through the hepatobiliary tract. Newly developed mouse models designed to study bilirubin metabolism have led to new insight into the role of the intestinal tract in controlling neonatal hyperbilirubinemia. Humanization of mice with the UGT1 locus (hUGT1 mice) and the UGT1A1 gene provide a unique tool to study the onset of hyperbilirubinemia since the human UGT1A1 gene is developmentally regulated during the neonatal period in hUGT1 mice. A new mechanism outlying developmental expression of intestinal UGT1A1 is presented and its implications in the control of neonatal hyperbilirubinemia discussed. New findings linking breast milk protection against necrotizing enterocolitis and intestinal control of UGT1A1 may help explain the contribution of breast milk toward the development of neonatal hyperbilirubinemia. Our findings outline a new model that includes an active intestinal ROS /IκB kinase/nuclear receptor corepressor 1 loop that can be applied to an understanding of breast milk-induced jaundice.

Fig. 1.

Bilirubin production and metabolism. Bilirubin is a byproduct of heme degradation when phagocytic cells, primarily macrophages, remove aged red blood cells (RBC) from the circulation. The production of biliverdin through heme oxygenase is the first major step, after which bilirubin is produced through biliverdin reductase. Bilirubin, on the other hand, is oxidized to biliverdin to form the biliverdin reductase cycle that functions as an important antioxidant in protecting lipid oxidation. Depending on the part of the body in which the breakdown occurs, such as Kupffer cells of the liver or cells in the spleen and bone marrow, the produced bilirubin eventually is released into the circulation, where bilirubin is taken up by plasma albumin protein and transported throughout the body. Once bilirubin reaches the liver, bilirubin enters the hepatocyte either passively or actively through OATP transporters. In the hepatocyte, bilirubin is metabolized through UGT1A1-catalyzed glucuronidation to form either mono- or di-glucuronides. The conjugates are secreted into bile via the multidrug resistance protein 2 (MRP2), and they in turn can be hydrolyzed by β-glucuronidase in the intestinal tract and then undergo enterohepatic circulation. Conventionally, liver is considered as the organ for bilirubin detoxification. It is worth noting that bilirubin is metabolized solely by UGT1A1, which becomes the rate limiting step. Therefore, insufficient UGT1A1 enzyme activity may lead to the rapid build-up of free bilirubin, a condition called hyperbilirubinemia or jaundice.

Fig. 2.

Humanized UGT1 mice and neonatal hyperbilirubinemia. Ugt1^−/− mice were generated by interrupting common exon 4 with a neomycin gene, impairing the function of the entire Ugt1 locus. Ugt1^−/− newborn mice are easily identified by their distinct orange skin color, which results from the accumulation of unconjugated bilirubin. The loss of the Ugt1 locus and UGT1A1 from the germ line is lethal, with all the Ugt1^−/− mice dying within 2 weeks after birth. When we crossed mice carrying the entire human UGT1 gene locus as a transgene with mice carrying the Ugt1-null allele, humanized UGT1 mice (hUGT1) were generated. The presence of the human UGT1A1 gene rescues the lethality associated with the absence of murine UGT1A1. All newborn hUGT1 mice develop severe neonatal hyperbilirubinemia (SNH). Total serum bilirubin (TSB) levels in hUGT1 newborns increase after birth, reach peak levels at approximately 2 weeks after birth with the levels returning to a normal range after weaning. The majority of the hUGT1 mice can mature to adulthood. Approximately 10% of newborn hUGT1 mice develop seizures and die. These mice accumulate high concentrations bilirubin in brain tissue. The development of SNH results from delayed liver UGT1A1 expression. UGT1A1 expression in the small intestine is associated with saving the lethality of hUGT1 mice and lowering of TSB levels.

Fig. 3.

Nuclear receptor (NR)-mediated activation, repression, and derepression of human UGT1A1 gene and the impact on hyperbilirubinemia in hUGT1 mice. Binding elements of various NRs, including PPARα, AhR, Nrf2, CAR, PXR, and LXRα, have been identified in the promoter region upstream of the transcription start site of the UGT1A1 gene. Upon ligand binding, these NRs can recruit coactivators and initiate downstream UGT1A1 gene expression. When hUGT1 neonates were exposed to different NR agonists, lowered serum bilirubin levels were observed (except for PPARγ). In the absence of ligand, some NRs associate with NR corepressors (such as NCoR1 and SMRT) and recruit chromatin-modifying enzyme HDACs to form a repressive complex and limit transcriptional activation. Disrupting of the repressive complex may lead to the derepression of gene expression. Humanized UGT1 mice deficient in PXR (hUGT/Pxr^−/− and hUGT1/Pxr^−/−/Car^−/−), but not CAR (hUGT1/Car^−/−), are associated with a reduction of total serum bilirubin. When selectively silenced, the corepressors SMRT, NCoR1, or HDACs in the primary hepatocytes isolated from hUGT1 mice, UGT1A1 gene activation was observed.

Fig. 4.

NCoR1 represses human UGT1 gene expression during development. NCoR1^F/F mice were crossed into hUGT1 to generate F/F^UN mice, which in turn were crossed with mice carrying Cre recombinase and knocked out NCoR1 specifically in liver (ΔHEP^UN) and intestinal tissue (ΔIEC^UN). Serum bilirubin levels were examined in neonatal mice, ΔHEP^UN mice exhibited a similar pattern of neonatal hyperbilirubinemia as hUGT1 (F/F^UN) mice, but this pattern was completely diminished with NCoR1 knockout in the intestinal tract of ΔIEC^UN mice. The change of bilirubin accumulation was also evidenced in fat tissues. Induction of intestinal UGT1A1 in ΔIEC^UN mice was observed and was most prominent throughout the developmental stage but did not occur after weaning or in adulthood. Data are expressed as mean ± SEM, **p<0.01, ***p<0.001, ****p<0.0001, Student's t test. Two-way ANOVA analysis for ΔIEC^UN vs F/F^UN (p<0.0001).

Fig. 5.

PEITC regulates UGT1A1 gene expression through ROS-mediated nuclear receptor signaling. Consumption of cruciferous vegetables leads to the generation of reactive oxygen species (ROS). ROS activates Nrf2 nuclear translocation, thus facilitating Nrf2-antioxidant response element (ARE) signaling and the transcriptional activation of the susceptible target genes. In addition, ROS are linked to the activation of CAR in both liver and small intestines, which results in the induction of Cyp2b10 and UGT1A1. The addition of N-acetylcysteine (NAC) blocks ROS production, nullifying both CAR and Nrf2 activation.

Fig. 6.

NCoR1 as a potential target for both neonatal hyperbilirubinemia and necrotizing enterocolitis (NEC). Intestinal UGT1A1 is essential in bilirubin detoxification and is directly linked to neonatal hyperbilirubinemia. We demonstrated that NCoR1 represses intestinal UGT1A1 gene expression in a developmental-dependent manner that is limited within the breastfeeding stage, suggesting that NCoR1 may function as an important underlying mechanism for BMJ. It is known that human milk oligosaccharides (HMOs) inhibit Toll-like receptors (TLRs), a family of membrane receptors upstream of NCoR1. When activated, TLRs activate IKKs and c-Jun signaling cascades, leading to turnover of NCoR1 from the target promoters and subsequent gene transcription. It hints at the possibility that inhibition of TLR function by HMOs leads to the heightened NCoR1 repression on the UGT1A1 gene, resulting in an increase in TSB levels. In addition, we now know that induction of intestinal UGT1A1 during the neonatal period by any means (i.e., oxidative stress, ligand-activated nuclear receptors, formula) will lower TSB values, adding support for a mechanism that implicates BM in facilitating reduced expression of UGT1A1. The establishment of the microflora starts immediately following birth. Pathogen-associated molecular patterns (PAMPs) can activate TLRs and induce an inflammatory response and gene transcription through NCoR1 derepression. This response may lead to important health concerns including necrotizing enterocolitis (NEC), a devastating disease that affects mostly premature infants. Countering this, premature infants on breast milk have demonstrated less NEC in comparison with formula feeding.