Liver Isoform Research Articles

Transient Hyperphosphatasemia in an Adolescent and an Adult Renal Transplant Patient

In renal transplant patients receiving immunosuppression, a significant increase in alkaline phosphatase (ALP) might be indicative of liver or bone diseases caused by many factors. In infancy and early childhood, a transient and therefore benign increase in ALP often has been described, usually during a course of infectious disease. Rarely, transient hyperphosphatasemia occurs in adults. We herein present 2 cases of transient hyperphosphatasemia in an adolescent and an adult renal transplant recipient, respectively. In the first case, a 17-year-old adolescent presented with an ALP value up to 2451 U/L, reporting no symptoms. In the second case, a 56-year-old woman with a second well-functioning kidney transplant presented with an ALP value up to 1532 U/L, without symptoms. In both cases, the biochemical profile and ultrasound study were negative for liver disease while no viral or other type of infection was detected. Bone scanning was within normal range and parathyroid hormone was also normal. However, bone ALP was measured at 8.9 and 11.9 times, respectively, above reference values. ALP electrophoresis had a characteristic pattern with involvement of both liver and bone-specific isoforms. About 6 weeks after their peak, ALP values gradually returned to normal range. Benign transient hyperphosphatasemia, although rare, should be considered in the differential diagnosis of isolated ALP increase, even in adult patients with kidney transplant. Electrophoresis of ALP could narrow the diagnostic procedure in cases when neither liver nor bone disease is clinically apparent.

Modulation of CYP2C9 activity and hydrogen peroxide production by cytochrome b5

Cytochromes P450 (CYP) play a major role in drug detoxification, and cytochrome b5 (cyt b5) stimulates the catalytic cycle of mono-oxygenation and detoxification reactions. Collateral reactions of this catalytic cycle can lead to a significant production of toxic reactive oxygen species (ROS). One of the most abundant CYP isoforms in the human liver is CYP2C9, which catalyzes the metabolic degradation of several drugs including nonsteroidal anti-inflammatory drugs. We studied modulation by microsomal membrane-bound and soluble cyt b5 of the hydroxylation of salicylic acid to gentisic acid and ROS release by CYP2C9 activity in human liver microsomes (HLMs) and by CYP2C9 baculosomes. CYP2C9 accounts for nearly 75% of salicylic acid hydroxylation in HLMs at concentrations reached after usual aspirin doses. The anti-cyt b5 antibody SC9513 largely inhibits the rate of salicylic acid hydroxylation by CYP2C9 in HLMs and CYP2C9 baculosomes, increasing the KM approximately threefold. Besides, soluble human recombinant cyt b5 stimulates the Vmax nearly twofold while it decreases nearly threefold the Km value in CYP2C9 baculosomes. Regarding NADPH-dependent ROS production, soluble recombinant cyt b5 is a potent inhibitor both in HLMs and in CYP2C9 baculosomes, with inhibition constants of 1.04 ± 0.25 and 0.53 ± 0.06 µM cyt b5, respectively. This study indicates that variability in cyt b5 might be a major factor underlying interindividual variability in the metabolism of CYP2C9 substrates.

Effects of Bardoxolone Methyl on Hepatic Enzymes in Patients with Type 2 Diabetes Mellitus and Stage 4 CKD

In a multinational placebo‐controlled phase III clinical trial in 2,185 patients with type 2 diabetes mellitus and stage 4 chronic kidney disease, treatment with the Nrf2 activator bardoxolone methyl increased estimated glomerular filtration rate, a measure of kidney function, but also resulted in increases in serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), and gamma glutamyl transferase. These increases in liver enzyme level(s) were maximal after 4 weeks of treatment and reversible, trending back toward baseline through week 48. Total bilirubin concentrations did not increase, and no cases met Hy’s Law criteria, although two subjects had ALT concentrations that exceeded 10 × the upper limit of the population reference range leading to discontinuation of treatment. Animal and cell culture experiments suggested that the increases in ALT and AST induced by bardoxolone methyl may be related to its pharmacological activity. Bardoxolone methyl significantly induced the mRNA expression of ALT and AST isoforms in cultured cells. Expression of ALT and AST isoforms in liver and kidney also positively correlated with Nrf2 status in mice. Overall, these data suggest that the increases in ALT and AST observed clinically were, at least in part, related to the pharmacological induction of aminotransferases via Nrf2 activation, rather than to any intrinsic form of hepatotoxicity.

Quantitative analysis of mRNA expression levels of aldo-keto reductase and short-chain dehydrogenase/reductase isoforms in human livers

The aldo-keto reductase (AKR) and short-chain dehydrogenase/reductase (SDR) superfamilies are responsible for the reduction in compounds containing the aldehyde, ketone, and quinone groups. In humans, 12 AKR isoforms (AKR1A1, AKR1B1, AKR1B10, AKR1B15, AKR1C1, AKR1C2, AKR1C3, AKR1C4, AKR1D1, AKR1E2, AKR7A2, and AKR7A3) and 6 SDR isoforms (CBR1, CBR3, CBR4, HSD11B1, DHRS4, and DCXR) have been found to catalyze the reduction in xenobiotics, but their hepatic expression levels are unclear. The purpose of this study is to determine the absolute mRNA expression levels of these 18 isoforms in the human liver. In 22 human livers, all isoforms, except for AKR1B15, are expressed, and AKR1C2 (on average 1.6 × 106 copy/μg total RNA), AKR1C3 (1.3 × 106), AKR1C1 (1.3 × 106), CBR1 (9.7 × 105), and HSD11B1 (1.1 × 106) are abundant, representing 67% of the total expression of reductases in the liver. The expression levels of AKR1C2, AKR1C3, AKR1C1, CBR1, and HSD11B1 are significantly correlated with each other, except between AKR1C2 and CBR1, suggesting that they might be regulated by common factor(s). In conclusion, this study comprehensively determined the absolute expression of mRNA expression of each AKR and SDR isoform in the human liver.

Affinity Crystallography Reveals Binding of Pomegranate Juice Anthocyanins at the Inhibitor Site of Glycogen Phosphorylase: The Contribution of a Sugar Moiety to Potency and Its Implications to the Binding Mode.

Anthocyanins (ACNs) are dietary phytochemicals with an acknowledged therapeutic significance. Pomegranate juice (PJ) is a rich source of ACNs with potential applications in nutraceutical development. Glycogen phosphorylase (GP) catalyzes the first step of glycogenolysis and is a molecular target for the development of antihyperglycemics. The inhibitory potential of the ACN fraction of PJ is assessed through a combination of in vitro assays, ex vivo investigation in hepatic cells, and X-ray crystallography studies. The ACN extract potently inhibits muscle and liver isoforms of GP. Affinity crystallography reveals the structural basis of inhibition through the binding of pelargonidin-3-O-glucoside at the GP inhibitor site. The glucopyranose moiety is revealed as a major determinant of potency as it promotes a structural binding mode different from that observed for other flavonoids. This inhibitory effect of the ACN scaffold and its binding mode at the GP inhibitor binding site may have significant implications for future structure-based drug design endeavors.

Abstract 240: Inhibition of glycogen degradation sensitizes glioblastoma cells to ionizing radiation

Abstract Glycogen storage has been demonstrated in multiple cancer cell lines and is upregulated in response to hypoxia both in vitro and in vivo. Evidence supports a potential role of the “glycogen shunt”, that describes the cycling of glucose through glycogen to subsequently fuel glycolysis, in brain tumors. Glioblastoma (GBM) is the most common and most deadly central nervous system tumor, and is characterized by hypoxia and metabolic reprogramming. We found that inhibition of glycogen degradation by downregulation of glycogen phosphorylase liver isoform (PYGL) in GBM cells led to a decrease in clonogenic growth and sensitization to ionizing radiation (IR) doses of 10-12 Gy. Control cells also showed growth impairment but resumed growth 10 days after IR exposure, while PYGL knockdown cells did not. To understand these different responses to high dose IR, we studied the intra-cellular effects of IR in control and PYGL knockdown GBM cells. Five days after IR exposure of PYGL knockdown cells, mitotic catastrophe and a senescence-like morphology were seen. Dysregulation of autophagy, accumulation of elongated and fragmented mitochondria, decrease in oxygen consumption rate and extracellular acidification rate, and increased pAMPK and pACC expression were shown in irradiated PYGL knockdown cells compared to control cells, suggesting metabolic stress. In human GBMs, PYGL mRNA expression was higher than in normal brain tissue and low PYGL mRNA expression was correlated with improved survival rates (hazard ratio 1.32, 95% confidence interval 1.08 - 1.61, P = 0.006). Immunohistochemical PYGL expression was also increased in human GBM versus normal brain tissue. In conclusion, inhibition of glycogen degradation sensitizes GBM cells to high dose IR, the cornerstone of GBM treatment, and may provide a way forward to improve treatment of primary GBMs. Citation Format: Anne M. Hendriks, Christos E. Zois, Syed B. Haider, Elisabete Pires, Dimitra Kalamida, Christoffer Lagerholm, John Morris, James S. McCullagh, Rudolf S. Fehrmann, Wilfred F. den Dunnen, Adrian L. Harris, Mathilde Jalving. Inhibition of glycogen degradation sensitizes glioblastoma cells to ionizing radiation [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 240.

Metabolism of sesamin by CYPs and UGTs in human liver

Sesamin is a major lignan in sesame, and its biological effects such as antioxidant effect, anti-carcinogenic effects, and suppression of hypertension have been extensively studied by many researchers. However, its metabolic pathways and metabolic enzymes in human bodies have not been identified. Recently we demonstrated that CYP2C9 was the most important cytochrome P450 isoform in human liver. Next, we focused on metabolism of sesamin mono-catechol by cytochrome P450 or UDP-glucuronosyltransferase (UGT). Further catecholization of sesamin mono-catechol by cytochrome P450 enhances its anti-oxidant activity, whereas glucuronidation by UGT strongly reduces anti-oxidant activity. In human liver microsomes, glucuronidation activity toward sesamin mono-catechol was much higher than the di-catecholization activity. In contrast, both activities were similar in rat liver microsomes. These results suggest a large species-based difference between humans and rats in sesamin metabolism. In vitro studies using 10 individual human liver microsomes suggested that UGT2B7 was responsible for glucuronidation of sesamin mono-catechol in human liver. In addition, we observed a significant methylation activity toward sesamin mono-catechol by catechol O-methyl transferase (COMT) in human liver cytosol. Based on these results, we concluded that CYP2C9, UGT2B7, and COMT played essential roles in the metabolism of sesamin in human liver.

Filament assembly by the liver phosphofructokinase‐1, the “gatekeeper” of glycolysis

Despite abundant knowledge of the regulation and biochemistry of glycolytic enzymes, we have limited understanding on how they are spatially organized in the cell. Emerging evidence indicates that metabolic enzymes from diverse pathways can assemble into filaments. We show that the liver isoform of glycolytic “gatekeeper” enzyme phosphofructokinase‐1 (PFKL), which catalyzes the step committing glucose to breakdown, forms filaments in vitro and dynamic punctae in cells. Recombinant PFKL, but not platelet or muscle isoforms, assembles into filaments in a concentration‐, substrate‐, and tetramer‐dependent manner. Filaments are apolar and made of stacked tetramers oriented with exposed catalytic sites positioned along the edge of the polymer as determined by negative‐stain electron micrographs. Quantified live‐cell imaging shows PFKL‐EGFP appears as dynamic puncta that are enriched at discrete locations at the plasma membrane. These findings reveal a new behavior of a key glycolytic enzyme with insights on spatial organization and isoform‐specific glucose metabolism in cells.Support or Funding InformationWest Virginia University Start up funds

Regulation of basal expression of hepatic PEPCK and G6Pase by AKT2.

Hepatic glucose metabolism signaling downstream of insulin can diverge to multiple pathways including AKT. Genetic studies suggest that AKT is necessary for insulin to suppress gluconeogenesis. To specifically address the role of AKT2, the dominant liver isoform of AKT in the regulation of gluconeogenesis genes, we generated hepatocytes lacking AKT2 (Akt2-/-). We found that, in the absence of insulin signal, AKT2 is required for maintaining the basal level expression of phosphoenolpyruvate carboxyl kinase (PEPCK) and to a lesser extent G6Pase, two key rate-limiting enzymes for gluconeogenesis that support glucose excursion due to pyruvate loading. We further showed that this function of AKT2 is mediated by the phosphorylation of cyclic AMP response element binding (CREB). Phosphorylation of CREB by AKT2 is needed for CREB to induce the expression of PEPCK and likely represents a priming event for unstimulated cells to poise to receive glucagon and other signals. The inhibition of gluconeogenesis by insulin is also dependent on the reduced FOXO1 transcriptional activity at the promoter of PEPCK. When insulin signal is absent, this activity appears to be inhibited by AKT2 in manner that is independent of its phosphorylation by AKT. Together, this action of AKT2 on FOXO1 and CREB to maintain basal gluconeogenesis activity may provide fine-tuning for insulin and glucocorticoid/glucagon to regulate gluconeogenesis in a timely manner to meet metabolic needs.

CPT1A-mediated Fat Oxidation, Mechanisms, and Therapeutic Potential.

Energy homeostasis during fasting or prolonged exercise depends on mitochondrial fatty acid oxidation (FAO). This pathway is crucial in many tissues with high energy demand and its disruption results in inborn FAO deficiencies. More than 15 FAO genetic defects have been currently described, and pathological variants described in circumpolar populations provide insights into its critical role in metabolism. The use of fatty acids as energy requires more than 2 dozen enzymes and transport proteins, which are involved in the activation and transport of fatty acids into the mitochondria. As the key rate-limiting enzyme of FAO, carnitine palmitoyltransferase I (CPT1) regulates FAO and facilitates adaptation to the environment, both in health and in disease, including cancer. The CPT1 family of proteins contains 3 isoforms: CPT1A, CPT1B, and CPT1C. This review focuses on CPT1A, the liver isoform that catalyzes the rate-limiting step of converting acyl-coenzyme As into acyl-carnitines, which can then cross membranes to get into the mitochondria. The regulation of CPT1A is complex and has several layers that involve genetic, epigenetic, physiological, and nutritional modulators. It is ubiquitously expressed in the body and associated with dire consequences linked with genetic mutations, metabolic disorders, and cancers. This makes CPT1A an attractive target for therapeutic interventions. This review discusses our current understanding of CPT1A expression, its role in heath and disease, and the potential for therapeutic opportunities targeting this enzyme.

Distinct Hepatic PKA and CDK Signaling Pathways Control Activity-Independent Pyruvate Kinase Phosphorylation and Hepatic Glucose Production

Pyruvate kinase is an important enzyme in glycolysis and a key metabolic control point. We recently observed a pyruvate kinase liver isoform (PKL) phosphorylation site at S113 that correlates with insulin resistance in rats on a 3day high-fat diet (HFD) and suggests additional control points for PKL activity. However, in contrast to the classical model of PKL regulation, neither authentically phosphorylated PKL at S12 nor S113 alone is sufficient to alter enzyme kinetics or structure. Instead, we show that cyclin-dependent kinases (CDKs) are activated by the HFD and responsible for PKL phosphorylation at position S113 in addition to other targets. These CDKs control PKL nuclear retention, alter cytosolic PKL activity, and ultimately influence glucose production. These results change our view of PKL regulation and highlight a previously unrecognized pathway of hepatic CDK activity and metabolic control points that may be important in insulin resistance and type 2 diabetes.

Expression of Cyp2c/Cyp2j subfamily members and oxylipin levels during LPS-induced inflammation and resolution in mice.

Inflammatory stimuli, such as bacterial LPS, alter the expression of many cytochromes P450. CYP2C and CYP2J subfamily members actively metabolize fatty acids to bioactive eicosanoids, which exhibit potent anti-inflammatory effects. Herein, we examined mRNA levels of the 15 mouse Cyp2c and 7 mouse Cyp2j isoforms in liver, kidney, duodenum, and brain over a 96-h time course of LPS-induced inflammation and resolution. Plasma and liver eicosanoid levels were also measured by liquid chromatography with tandem mass spectrometry. Expression changes in Cyp2c and Cyp2j isoforms were both isoform and tissue specific. Total liver Cyp2c and Cyp2j mRNA content was reduced by 80% 24 h after LPS but recovered to baseline levels by 96 h. Total Cyp2c and Cyp2j mRNA in kidney (-19%) and duodenum (-64%) were reduced 24 h after LPS but recovered above baseline by 72 h. Total Cyp2c and Cyp2j mRNA content in brain was elevated at all time points after LPS dosing. Plasma eicosanoids transiently increased 3-6 h after administration of LPS. In liver, esterified oxylipin levels decreased during acute inflammation and before recovering. The biphasic suppression and recovery of mouse Cyp2c and Cyp2j isoforms and associated changes in eicosanoid levels during LPS-induced inflammation and resolution may have important physiologic consequences.-Graves, J. P., Bradbury, J. A., Gruzdev, A., Li, H., Duval, C., Lih, F. B., Edin, M. L., Zeldin, D. C. Expression of Cyp2c/Cyp2j subfamily members and oxylipin levels during LPS-induced inflammation and resolution in mice.

Fatty acids modulate the expression of pyruvate kinase and arachidonate-lipoxygenase through PPARγ/CYP2C45 pathway: a link to goose fatty liver

Cytochrome P-450 2C45 (CYP2C45) is the most highly expressed cytochrome P-450 isoform in chicken liver, and may play an important role in avian liver biology. However, information regarding the function of CYP2C45 in fatty liver is generally limited. The aim of this study was to investigate the role of CYP2C45 during the development of goose fatty liver. Our result indicated that the transcription of CYP2C45, together with PK and ALOX5, was increased in goose liver upon overfeeding for 19 D (P < 0.05). In goose primary hepatocytes, CYP2C45 RNA expression was also upgraded by the treatment with various chemicals like insulin, the fatty acids, and PPAR agonists (P < 0.05). We also found that both CYP2C45 overexpression and troglitazone treatment could increase the expression of pyruvate kinase (PK) and arachidonate 5-lipoxygenase (ALOX5), and furthermore, showed that the up-regulation of PK and ALOX5 induced by troglitazone could be suppressed by small interfering RNAs targeting CYP2C45 (P < 0.05). These findings suggest that fatty acids treatment and the overfeeding can induce the up-regulation of CYP2C45 expression possibly via PPARγ and that the induction of PK and ALOX5 in goose fatty liver is at least partially attributed to fatty acid-induced expression of CYP2C45. Thus, our data provides an insight into the mechanism by which glycolysis and arachidonic acid metabolism are modulated in goose fatty liver.

Breast cancers utilize hypoxic glycogen stores via PYGB, the brain isoform of glycogen phosphorylase, to promote metastatic phenotypes.

In breast cancer, tumor hypoxia has been linked to poor prognosis and increased metastasis. Hypoxia activates transcriptional programs in cancer cells that lead to increased motility and invasion, as well as various metabolic changes. One of these metabolic changes, an increase in glycogen metabolism, has been further associated with protection from reactive oxygen species damage that may lead to premature senescence. Here we report that breast cancer cells significantly increase glycogen stores in response to hypoxia. We found that knockdown of the brain isoform of an enzyme that catalyzes glycogen breakdown, glycogen phosphorylase B (PYGB), but not the liver isoform, PYGL, inhibited glycogen utilization in estrogen receptor negative and positive breast cancer cells; whereas both independently inhibited glycogen utilization in the normal-like breast epithelial cell line MCF-10A. Functionally, PYGB knockdown and the resulting inhibition of glycogen utilization resulted in significantly decreased wound-healing capability in MCF-7 cells and a decrease in invasive potential of MDA-MB-231 cells. Thus, we identify PYGB as a novel metabolic target with potential applications in the management and/or prevention of metastasis in breast cancer.

Role of hepatocyte nuclear factor 4-alpha in gastrointestinal and liver diseases.

Hepatocyte nuclear factor 4-alpha (HNF4α) is a highly conserved member of nuclear receptor superfamily of ligand-dependent transcription factors that is expressed in liver and gastrointestinal organs (pancreas, stomach, and intestine). In liver, HNF4α is best known for its role as a master regulator of liver-specific gene expression and essential for adult and fetal liver function. Dysregulation of HNF4α expression has been associated with many human diseases such as ulcerative colitis, colon cancer, maturity-onset diabetes of the young, liver cirrhosis, and hepatocellular carcinoma. However, the precise role of HNF4α in the etiology of these human pathogenesis is not well understood. Limited information is known about the role of HNF4α isoforms in liver and gastrointestinal disease progression. There is, therefore, a critical need to know how disruption of the expression of these isoforms may impact on disease progression and phenotypes. In this review, we will update our current understanding on the role of HNF4α in human liver and gastrointestinal diseases. We further provide additional information on possible use of HNF4α as a target for potential therapeutic approaches.

Tissue specific alpha-2-Macroglobulin (A2M) splice isoform diversity in Hilsa shad, Tenualosa ilisha (Hamilton, 1822).

The present study, for the first time, reported twelve A2M isoforms in Tenualosa ilisha, through SMRT sequencing. Hilsa shad, T. ilisha, an anadromous fish, faces environmental stresses and is thus prone to diseases. Here, expression profiles of different A2M isoforms in four tissues were studied in T. ilisha, for the tissue specific diversity of A2M. Large scale high quality full length transcripts (>0.99% accuracy) were obtained from liver, ovary, testes and gill transcriptomes, through Iso-sequencing on PacBio RSII. A total of 12 isoforms, with complete putatative proteins, were detected in three tissues (7 isoforms in liver, 4 in ovary and 1 in testes). Complete structure of A2M mRNA was predicted from these isoforms, containing 4680 bp sequence, 35 exons and 1508 amino acids. With Homo sapiens A2M as reference, six functional domains (A2M_N,A2M_N2, A2M, Thiol-ester_cl, Complement and Receptor domain), along with a bait region, were predicted in A2M consensus protein. A total of 35 splice sites were identified in T. ilisha A2M consensus transcript, with highest frequency (55.7%) of GT-AG splice sites, as compared to that of Homo sapiens. Liver showed longest isoform (X1) consisting of all domains, while smallest (X10) was found in ovary with one Receptor domain. Present study predicted five putative markers (I-212, I-269, A-472, S-567 and Y-906) for EUS disease resistance in A2M protein, which were present in MG2 domains (A2M_N and A2M_N2), by comparing with that of resistant and susceptible/unknown response species. These markers classified fishes into two groups, resistant and susceptible response. Potential markers, predicted in T. ilisha, placed it to be EUS susceptible category. Putative markers reported in A2M protein may serve as molecular markers in diagnosis of EUS disease resistance/susceptibility in fishes and may have a potential for inclusion in the marker panel for pilot studies. Further, challenging studies are required to confirm the role of particular A2M isoforms and markers identified in immune protection against EUS disease.

AMP-activated protein kinase complexes containing the β2 regulatory subunit are up-regulated during and contribute to adipogenesis.

AMP-activated protein kinase (AMPK) is a heterotrimer of α-catalytic and β- and γ-regulatory subunits that acts to regulate cellular and whole-body nutrient metabolism. The key role of AMPK in sensing energy status has led to significant interest in AMPK as a therapeutic target for dysfunctional metabolism in type 2 diabetes, insulin resistance and obesity. Despite the actions of AMPK in the liver and skeletal muscle being extensively studied, the role of AMPK in adipose tissue and adipocytes remains less well characterised. Small molecules that selectively influence AMPK heterotrimers containing specific AMPKβ subunit isoforms have been developed, including MT47-100, which selectively inhibits complexes containing AMPKβ2. AMPKβ1 and AMPKβ2 are the principal AMPKβ subunit isoforms in rodent liver and skeletal muscle, respectively, yet the contribution of specific AMPKβ isoforms to adipose tissue function, however, remains largely unknown. This study therefore sought to determine the contribution of AMPKβ subunit isoforms to adipocyte biology, focussing on adipogenesis. AMPKβ2 was the principal AMPKβ isoform in 3T3-L1 adipocytes, isolated rodent adipocytes and human subcutaneous adipose tissue, as assessed by the contribution to total cellular AMPK activity. Down-regulation of AMPKβ2 with siRNA inhibited lipid accumulation, cellular adiponectin levels and adiponectin secretion during 3T3-L1 adipogenesis, whereas down-regulation of AMPKβ1 had no effect. Incubation of 3T3-L1 cells with MT47-100 selectively inhibited AMPK complexes containing AMPKβ2 whilst simultaneously inhibiting cellular lipid accumulation as well as cellular levels and secretion of adiponectin. Taken together, these data indicate that increased expression of AMPKβ2 is an important feature of efficient adipogenesis.

Development and validation of an absolute protein assay for the simultaneous quantification of fourteen CYP450s in human microsomes by HPLC-MS/MS-based targeted proteomics

The cytochrome P450 (CYP450) superfamily constitutes the major enzymatic system involved in drug metabolism. CYP450s are highly expressed in the liver and other tissues and limited data on absolute characterization of CYP450s in extra hepatic organs, such as the small intestine, are available. Our objective was to develop and validate an absolute quantification assay by HPLC-MS/MS-based targeted proteomics allowing the simultaneous quantification of fourteen major human CYP450 isoforms (CYP1A1, 1A2, 1B1, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, 2J2, 3A4, 3A5, 3A7 and 4F2) in human liver and intestine microsomes. Absolute protein quantification was performed using two proteotypic peptides for each of the fourteen CYP450s. Peptides were obtained after a tryptic digestion of microsomes and samples were analyzed by high performance liquid chromatography with heated electrospray ionization tandem mass spectrometry (HPLC-HESI-MS/MS). Chromatographic separation was performed on a Biobasic-8 analytical column (5 μm 100 x 1 mm) with a gradient elution using acetonitrile and water both fortified with 0.1% formic acid (flow rate: 75 μL/min). Calibration curves were linear over a wide range of concentrations (0.1–50 nM) and the assay met all requirements of sensitivity, linearity, precision, accuracy and matrix effect. Strong correlations were observed between the two proteotypic peptides for each isoenzyme, corroborating the strength of this method. Twelve CYP450s were detected in commercially available human liver microsomes while seven CYP450s were detected in human intestine microsomes. To our knowledge, this is the most sensitive (0.1 nM) and the first most extensively validated assay that can be applied to the absolute quantification of CYP450s in human tissues.

SUN-023 HNF4a Isoforms Regulate the Circadian Balance between Carbohydrate and Fatty Acid Metabolism in the Liver

Nuclear receptor genes often contain alternative promoters but the physiological relevance of the different promoters or the proteins they generate is largely unknown. Hepatocyte Nuclear Factor 4α (HNF4α), master regulator of hepatocyte differentiation and the most abundant nuclear receptor in the liver, is regulated by two promoters (P1 and P2). P1-HNF4α, a tumor suppressor, is the major isoform in the adult liver while P2-HNF4α is expressed in fetal liver and liver cancer: a role for P2-HNF4α in normal adult liver has not been identified. Here we show for the first time that P2-HNF4α is expressed at ZT9 and ZT21 in the normal adult liver and use P2-HNF4α-expressing exon swap mice and ‘omics approaches to demonstrate that P2-HNF4α orchestrates a distinct transcriptome and metabolome via unique chromatin and protein-protein interactions. Cytochrome P450 and sex-specific gene expression is impacted as well as carbohydrate and fatty acid metabolism. The exon swap mice exhibit subtle differences in circadian gene regulation and disruption of the clock increases expression of P2-HNF4α. They also exhibit notable differences in carbohydrate versus fatty acid metabolism with P1-HNF4α driving gluconeogenesis and P2-HNF4α driving ketogenesis. Our results also show that P2-HNF4α is required for the elevated levels of ketone bodies in female mice. Taken together, we propose that the highly conserved two-promoter structure in the Hnfa gene is designed, at least in part, to respond to the availability of food resources and maintain the balance between gluconeogenesis and ketogenesis in a circadian and sex-specific fashion. (Funding: NIH NIDDK, West Cost Metabolomics Center, NIEHS T32, USDA NIFA, CCFA, Superfund Research Program)

Comprehensive characterization of mouse hepatic UDP‐glucuronosyltransferases: Major participation of Ugt2b1 in bisphenol A glucuronidation

PurposeBisphenol A (BPA), which exhibits estrogen‐like properties, is a starting material for the synthesis of plastic especially for polycarbonates. The endocrine disruption due to the contamination of BPA in the baby bottle has been of much concern. The mouse has been regarded as an important animal for endocrinological and toxicological studies. Although UGT2B1 is known as one of the catalysts in rat liver, the isoforms participating in BPA glucuronidation in mouse have not yet been identified. To address this issue, we comprehensively characterized all the Ugt isoforms in mouse liver for BPA glucuronidation.MethodscDNAs of all the Ugt isoforms in mouse liver were amplified from total RNA of FVB mouse liver and cloned into pFastBac1. A baculovirus‐Sf9 cell system for expressing each Ugt isoform was established. The activity of microsomes expressing recombinant mouse Ugt was determined using the substrate BPA by ultra performance liquid chromatography with UV detection.Results and DiscussionOn the basis of the kinetic parameters of BPA glucuronidation by mouse liver microsomes, substrate concentrations were set at 100 μM (near Km) and 400 μM, which is expected to give Vmax. Of all the Ugt mouse‐liver isoforms examined, Ugt2b1 had the highest activity toward BPA at 100 μM while much lower activities were observed with Ugt2b34, Ugt1a9, and Ugt1a6b. Furthermore, Ugt2b1 consistently exhibited high activity toward BPA at 400 μM whereas the activity of Ugt2b34, Ugt1a9 and Ugt1a6b was lowered at this high concentration. The other isoforms exhibited little activity toward BPA at 100 μM and 400 μM.ConclusionsThese results suggest that Ugt2b1 is the major contributor to BPA glucuronidation in mouse liver. To address the glucuronidation of BPA at the level which causes endocrine disruption, the major participation of Ugt2b1 followed by Ugt2b34, Ugt1a9 and Ugt1a6b should be considered.Support or Funding InformationJSPS KAKENHI Grant Number JP17H00788This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.