Result of a Pilot External Quality Assessment Scheme for Clinical Diagnosis of Inherited Metabolic Disorders in China.

BACKGROUND: We aimed to evaluate the diagnostic capabilities of Chinese laboratories for inherited metabolic disorders (IMDs) using gas chromatography-mass spectrometry (GC-MS) on urine samples. Meanwhile, based on the result of the pilot external quality assessment (EQA) scheme, we hope to establish a standardized and reliable procedure for future EQA practice. METHODS: We recruited laboratories that participated in the EQA of quantitative analysis of urinary organic acids with GC-MS before joining the surveys. In each survey, a set of five real urine samples was distributed to each participant. The participants should analyze the sample by GC-MS and report the "analytical result", "the most likely diagnosis", and "recommendation for further tests" to the NCCL before the deadline. RESULTS: A total of 21 laboratories participated in the scheme. The pass rates were 94.4% in 2020 and 89.5% in 2021. For all eight IMDs tested, the analytical proficiency rates ranged from 84.7% - 100%, and the interpretational performance rate ranged from 88.2% - 97.0%. The performance on hyperphenylalaninemia (HPA), 3-methylcrotonyl-CoA carboxylase deficiency (MCCD), and ethylmalonic encephalopathy (EE) samples were not satisfactory. CONCLUSIONS: In general, the participants of this pilot EQA scheme are equipped with the basic capability for qualitative organic acid analysis and interpretation of the results. Limited by the small size of laboratories and samples involved, this activity could not fully reflect the state of clinical practice of Chinese laboratories. NCCL will improve the EQA scheme and implement more EQA activities in the future.

[Prenatal genetic diagnosis of a case with ring chromosome 13].

OBJECTIVE: To carry out cyto- and molecular genetic analysis for a fetus with a ring chromosome identified through non-invasive prenatal testing (NIPT). METHODS: A pregnant woman presented at the Shengjing Hospital Affiliated to China Medical University on May 11, 2021 was selected as the study subject. Maternal peripheral blood sample was screened by NIPT, and G-banded chromosomal karyotyping was carried out on amniotic fluid and peripheral blood samples from the couple. The fetus and the pregnant woman were also subjected to genomic copy number variation sequencing (CNV-seq), chromosomal microarray analysis (CMA), and fluorescence in situ hybridization (FISH) assay. RESULTS: NIPT result suggested that the fetus had monomeric mosaicism or fragment deletion on chromosome 13. G banded chromosomal analysis showed that both the fetus and its mother had a karyotype of 47,XX,der(13)(pter→p11::q22→q10),+r(13)(::p10::q22→qter::), whilst her husband had a normal karyotype. FISH has verified the above results. No abnormality was detected with CNV-seq and CMA in both the fetus and the pregnant woman. CONCLUSION: The ring chromosome 13 in the fetus has derived from its mother without any deletion, duplication and mosaicism. Both the fetus and the pregnant woman were phenotypically normal.

A novel variant in the FBP1 gene causes fructose-1,6-bisphosphatase deficiency through increased ubiquitination.

Fructose-1,6-bisphosphatase (FBPase) deficiency is an autosomal recessive disorder characterized by impaired gluconeogenesis caused by mutations in the fructose-1,6-bisphosphatase 1 (FBP1) gene. The molecular mechanisms underlying FBPase deficiency caused by FBP1 mutations require investigation. Herein, we report the case of a Chinese boy with FBPase deficiency who presented with hypoglycemia, ketonuria, metabolic acidosis, and repeated episodes of generalized seizures that progressed to epileptic encephalopathy. Whole-exome sequencing revealed compound heterozygous variants, c.761 A > G (H254R) and c.962C > T (S321F), in FBP1. The variants, especially the novel H254R, reduced protein stability and enzymatic activity in patient-derived leukocytes and transfected HepG2 and U251 cells. Mutant FBP1 undergoes enhanced ubiquitination and proteasomal degradation. NEDD4-2 was identified as an E3 ligase for FBP1 ubiquitination in transfected cells and the liver and brain of Nedd4-2 knockout mice. The H254R mutant FBP1 interacted with NEDD4-2 at significantly higher levels than the wild-type control. Our study identified a novel H254R variant of FBP1 underlying FBPase deficiency and elucidated the molecular mechanism underlying the enhanced NEDD4-2-mediated ubiquitination and proteasomal degradation of mutant FBP1.

Discrepancy between non-invasive prenatal testing result and fetal karyotype caused by rare confined placental mosaicism: A case report.

BACKGROUND: Confined placental mosaicism (CPM) is one of the major reasons for discrepancies between the results of non-invasive prenatal testing (NIPT) and fetal karyotype analysis. CASE SUMMARY: We encountered a primiparous singleton pregnant woman with a rare CPM consisting of 47,XY,+21; 47,XXY; and 46,XY, who obtained a false-positive result on NIPT with a high risk for trisomy 21. Copy-number variation sequencing on amniotic fluid cells, fetal tissue, and placental biopsies showed that the fetal karyotype was 47,XXY, while the placenta was a rare mosaic of 47,XY,+21; 47,XXY; and 46,XY. CONCLUSION: The patient had a rare CPM consisting of 47,XY,+21; 47,XXY; and 46,XY, which caused a discrepancy between the result of NIPT and the actual fetal karyotype. It is important to remember that NIPT is a screening test, not a diagnostic test. Any positive result should be confirmed with invasive testing, and routine ultrasound examination is still necessary after a negative result.

X-linked adrenoleukodystrophy caused by maternal ABCD1 mutation and paternal X chromosome inactivation.

X-linked adrenoleukodystrophy (X-ALD) is the most common peroxisomal disorder. It is caused by defects in the ATP-binding cassette subfamily D member 1 (ABCD1) gene, resulting in impaired peroxisomal ß-oxidation of very-long-chain fatty acids (VLCFAs). As an X-linked recessive disease, female X-ALD carriers are typically asymptomatic. In the present study, a 7-year-old girl was diagnosed with cerebral ALD. Brain magnetic resonance imaging revealed asymmetric demyelination of bilateral white matter. Plasma VLCFAs level showed a substantial increase. Whole exome and Sanger sequencing revealed an ABCD1 c.919C>T (p.Q307X) heterozygous pathogenic mutation, which was inherited from the asymptomatic mother. X chromosome inactivation (XCI) analysis revealed that the normal paternal X chromosome was almost completely inactivated. Thus, the maternal ABCD1 mutation and paternal XCI were responsible for causing the disease in the patient. XCI may be one reason female X-ALD carriers can be symptomatic.

Renal NKCC2 Is Dual Regulated by the Synergy of 20-HETE and High-Fat Diet in CYP4F2 Transgenic Mice.

INTRODUCTION: 20-Hydroxyeicosatetraenoic acid (20-HETE) is the metabolite of cytochrome P450, which modulates blood pressure by inhibiting renal sodium transport. However, the molecular mechanisms underlying the role of 20-HETE in the development of obesity-related hypertension remain unclear, necessitating this study. METHODS: Cytochrome P450 4F2 (CYP4F2) transgenic mice fed high-fat diet (HFD) were used as research animal models. The expression of renal ion transport molecules targeted by 20-HETE was evaluated by real-time PCR and Western blot (WB). The regulatory effect of 20-HETE and HFD on renal Na+-K+-2Cl- cotransporter, isoform 2 (NKCC2) was explored by immunoprecipitation, WB, and luciferase assay. RESULTS: A 2-week HFD feeding dramatically decreased protein abundance but increased renal NKCC2 mRNA expression in CYP4F2 transgenic mice. The decrease in NKCC2 protein was demonstrated to be due to ubiquitination induced by the synergy between 20-HETE and HFD. The increased PPAR-γ protein in CYP4F2 transgenic mice fed HFD and the activation of rosiglitazone on the luciferase reporter construct of the NKCC2 promoter demonstrated that the increase in NKCC2 mRNA in CYP4F2 transgenic mice fed HFD was a consequence of elevated PPAR-γ protein induced by the synergy between 20-HETE and HFD. CONCLUSIONS: Our data demonstrated that the synergy between 20-HETE and HFD could decrease NKCC2 protein via posttranslational ubiquitination, which was thought to be the main mechanism underlying the short-term effect in response to HFD and might be responsible for the adaptive modulation of renal NKCC2 to resist sodium retention. Moreover, the increased NKCC2 mRNA expression via PPAR-γ-induced transcriptional regulation was thought to be the main mechanism underlying the long-term effect in response to HFD and plays a pivotal role in the development of obesity-related hypertension.

Crosstalk between phosphorylation and ubiquitination is involved in high salt-induced WNK4 expression.

With no lysine 4 (WNK4) is a serine/threonine kinase, which is expressed in the kidney and associated with salt-sensitive hypertension. However, how salt regulates WNK4 remains unclear. In the present study, the C57BL/6 mice and HEK293 cells were treated with high salt and the expression of WNK4 protein and its ubiquitination and phosphorylation levels were detected. Western blotting demonstrated that WNK4 expression was significantly increased in high salt-treated mice and cells. Meanwhile, co-immunoprecipitation analysis demonstrated that the ubiquitination of WNK4 was decreased under high-salt simulation. It was also identified that the Lys-1023 site was the most important ubiquitination site for WNK4, and it was found that phosphorylation at the Ser-1022 site was a prerequisite for ubiquitination. These results suggested that there was crosstalk between phosphorylation and ubiquitination in the WNK4 protein, and high salt may downregulate its phosphorylation and, in turn, decrease its ubiquitination, leading to a decrease in WNK4 degradation. This eventually resulted in an increase in the abundance of WNK4 protein.

Homocysteine downregulates cardiac homeobox transcription factor NKX2.5 via IGFBP5.

Homocysteine (Hcy) is an independent risk factor of congenital heart disease (CHD), but its exact underlying mechanism is unclear. In this study, we collected amniotic fluid (AF) supernatant samples from pregnant women carrying CHD-affected (n = 16) or normal (n = 16) fetuses. We found that Hcy concentrations were higher in the AF of the CHD group when compared with normal pregnancies. Also, Western blot showed that NK2 homeobox 5 (NKX2.5) was decreased and insulin-like growth factor binding protein 5 (IGFBP5) was increased in the AF of the CHD group. In the H9C2 cell culture experiment, 500 µmol/L Hcy downregulated NKX2.5 and upregulated IGFBP5. Real-time PCR and Western blot showed that NKX2.5 expression was reduced in H9C2 cells treated with IGFBP5. Luciferase reporter gene demonstrated that IGFBP5 decreased the transcription of the NKX2.5 promoter. Chromatin immunoprecipitation and electrophoretic mobility shift assay suggested that IGFBP5 binds to the NKX2.5 promoter region. Thus, the data indicated that one of the possible mechanisms by which Hcy is involved in CHD may be that Hcy inhibits NKX2.5 expression partly through IGFBP5.NEW & NOTEWORTHY We found that Hcy and IGFBP5 were increased, whereas NKX2.5 was decreased, in AF of CHD. Meanwhile, Hcy could upregulate IGFBP5 but downregulate NKX2.5, and IGFBP5 inhibited NKX2.5 expression in vitro. Moreover, IGFBP5 can bind to the NKX2.5 promoter region and reduce NKX2.5 transcriptional activity.

Role of Nkx2.5 in H2O2-induced Nsd1 suppression.

Nuclear receptor-binding SET domain-containing protein 1 (Nsd1) acts as a histone lysine methyltransferase, and its role in oxidative stress-related abnormal embryonic heart development remains poorly understood. In the present study, H2O2 decreased the expression of Nsd1 and NK2 transcription factor related locus 5 (Nkx2.5). We further focused on Nkx2.5 modulating the transcription of Nsd1 in response to H2O2. Luciferase activity analysis indicated that a regulatory region from - 646 to - 282 is essential for the basal transcriptional activity, in which, an a Nkx2.5-binding element (NKE) was identified at - 412/- 406 of the Nsd1 promoter by electrophoresis mobility shift assay and a chromatin immunoprecipitation assay. H2O2 obviously reduced the p646-luc promoter activity, and the depletion of Nkx2.5 expression weakened H2O2 inhibition on the p646-luc promoter. The overexpression of Nkx2.5 increase Nsd1 p646-luc promoter activity, but did not affected p646-luc-mut. Furthermore, overexpression and depletion of Nkx2.5 led to the increase and decrease of Nsd1 protein and mRNA levels. These data indicated that H2O2-induced Nsd1 suppression resulted from the decrease of Nkx2.5 expression through the NKE element.

Decreased Amino Acid Concentrations are Involved in Congenital Heart Disease.

OBJECTIVE: Congenital heart disease (CHD) is the most common malformation in China. In this study, we determined whether amino acids (AAs) in the amniotic fluid (AF) of patients with CHD changed and clarified whether AAs would affect the insulin-like growth factor type 1 receptor (IGF1R). METHOD: Fifty-seven AF samples from pregnant women carrying CHD-affected (n = 17) or normal (n = 40) fetuses were collected. The AA concentrations were measured in AF by liquid chromatography-tandem mass spectrometry. The IGF axis-related and epigenetic marker proteins in AF after serial treatments were quantified using a multiple reaction monitoring approach. IGF1R and P300 were also confirmed by Western blot in AF without any treatment. RESULTS: Most AAs decreased in the AF of patients with CHD. P300 and IGF1R decreased significantly in the CHD group. When H9C2 cells were cultured in one-half AA concentrations, the expression of P300 and IGF1R was reduced. Histone acetylation of the IGF1R promoter also decreased. CONCLUSION: Our data suggest that AAs decreased in the AF of patients with CHD. AAs may partly regulate the IGF1R through P300, which may be involved in heart development.

20-HETE regulated PSMB5 expression via TGF-ß/Smad signaling pathway.

We previously found that 20-hydroxyeicosatetraeonic acid (20-HETE) showed an effect on proteasome activity in cytochrome P450 F2 (CYP4F2) transgenic mice. Proteasome subunit ß5 (PSMB5) is a primary subunit of the proteasome. In the current study, we examine whether 20-HETE has any affect on PSMB5. We found that PSMB5 was upregulated in the liver, but downregulated in the kidney of transgenic mice, when compared with wild-type mice. Luciferase reporter gene experiments and electrophoretic mobility shift assays (EMSA) suggested that Smad3 directly associated with the putative Smad binding element (SBE) of the Psmb5 promoter. Furthermore, the binding affinity was different between the liver and kidney, and can be regulated by 20-HETE. Compared to wild mice, both TGF-ß1 and Smad3 phosphorylation were increased in the liver but decreased in the kidney of transgenic mice. SB431542, an inhibitor of TGF-ß receptor I kinase activity, can reverse the changes induced in PSMB5 by 20-HETE in vitro. Taken together, our data demonstrated that 20-HETE upregulated the expression of PSMB5 by activating the TGF-ß/Smad signaling pathway in the liver, but downregulated the expression of PSMB5 by inhibiting the TGF-ß/Smad signaling pathway in the kidney of transgenic mice.

20-Hydroxyeicosatetraenoic acid regulates the expression of Nedd4­2 in kidney and liver through a neddylation modification pathway.

The present study aimed to test whether 20-hydroxyeicosatetraenoic acid (20­HETE) affected neddylation modification of E3­ligase Nedd4­2 (neural precursor cell expressed, developmentally down­regulated 4­like, E3 ubiquitin protein ligase). A cytochrome P450 family 4 subfamily F member 2 (CYP4F2) transgenic mouse model that overproduces 20­HETE in the kidney and the liver was used in the present study. Transgenic mice with high salt intake exhibited increased activation of Nedd4­2­mediated ubiquitin­proteasome pathway. Nedd4­2 expression is increased in the kidney and decreased in the liver of transgenic mice compared with wild­type mice. Subsequently, co­immunoprecipitation analysis indicated that Nedd4­2 was modified by Nedd8, and the level of neddylation on Nedd4­2 was reduced in the kidney and increased in the liver of transgenic mice compared with controls. In addition, sentrin­specific protease 8 (Senp8), a deneddylation enzyme, is expressed higher in the kidney and lower in the liver of transgenic mice compared with wild­type controls. The function of 20­HETE on modulation of Nedd4­2 were also confirmed in mouse M1 kidney and mouse NCTC1469 liver cell lines, and the function was restored by neddylation inhibitor MLN4924 Data from the present study demonstrated that 20­HETE upregulated the expression of Nedd4­2 in the kidney and downregulated expression in the liver through the neddylation modification pathway, at least partly, depending on the effects on Senp8 deneddylation.

miR-137 downregulates c-kit expression in acute myeloid leukemia.

The oncogene c-kit plays a vital role in the pathogenesis of acute myeloid leukemia (AML). However, the mechanism of microRNAs targeting c-kit in AML has not been determined in detail. Moreover, the role miR-137 in tumor cell proliferation remains controversial. The aim of this work was to verify whether miR-137 targets c-kit and to research the biological effects of restoring miR-137 expression in leukemia cells. We found that miR-137 binds specifically to the 3'-UTR of c-kit and suppresses the expression and activities of c-kit. There is a negative correlation between miR-137 and c-kit expression in both patients and cell lines determined by screening large clinical samples. We found that miR-137 can inhibit proliferation, promote apoptosis, and induce differentiation of c-kit+ AML cells. We determined that miR-137 can participate in the leukemogenesis by regulating c-kit, which could be used as a therapeutic target for acute myeloid leukemia.

cMyBP-C was decreased via KLHL3-mediated proteasomal degradation in congenital heart diseases.

Cardiac myosin binding protein C (cMyBP-C) is a cardiac structural and regulatory protein; mutations of cMyBP-C are frequently associated with hypertrophic cardiomyopathy (HCM) and dilated cardiomyopathy (DCM). Cardiac special transcription factors may regulate the expression of cMyBP-C. However, the role of cMyBP-C in congenital heart diseases (CHD) remains poorly understood. In the current study, western blotting and the MRM approach showed that cMyBP-C expression was significantly reduced in fetuses with CHD compared to those without. Furthermore, we found that cMyBP-C interacted with KLHL3 by immunoprecipitation and immunofluorescence, and the degradation of cMyBP-C was caused by KLHL3-mediated ubiquitination. In addition, homocysteine (Hcy, a risk factor of CHD) treatment caused a decrease in cMyBP-C and an increase in KLHL3 expression, and the proteasome inhibitor MG132 reversed the Hcy-induced reduction of cMyBP-C expression. Finally, we verified that reduced cMyBP-C by Hcy promoted apoptosis in cardiomyocytes. These results demonstrate that Hcy decreases the expression of cMyBP-C through a KLHL3-mediated ubiquitin-proteasome pathway, and thereby influences heart development.

Alteration of epoxyeicosatrienoic acids in the liver and kidney of cytochrome P450 4F2 transgenic mice.

Arachidonic acid (AA) can be metabolized into 20-hydroxyeicosatetraenoic acid (20-HETE) by ω-hydroxylases, and epoxyeicosatrienoic acids (EETs) by epoxygenases. The effects of EETs in cardiovascular physiology are vasodilatory, anti-inflammatory and anti­apoptotic, which are opposite to the function to 20­HETE. However, EETs are not stable in vivo, and are rapidly degraded to the biologically less active metabolites, dihydroxyeicosatrienoic acids, via soluble epoxide hydrolase (sEH). Western blotting, reverse transcription­quantitative polymerase chain reaction and liquid chromatography tandem mass spectrometry were performed in order to determine target RNA and protein expression levels. In the present study, it was demonstrated that the disturbed renal 20­HETE/EET ratio in the hypertensive cytochrome P450 4F2 transgenic mice was caused by the activation of sEH and the repression of epoxygenase activity. In addition, 20­HETE showed an opposite regulatory effect on the endogenous epoxygenases in the liver and kidney. Given that 20­HETE and EETs have opposite effects in multiple disease, the regulation of their formation and degradation may yield therapeutic benefits.

20-HETE attenuates the response of glucose-stimulated insulin secretion through the AKT/GSK-3ß/Glut2 pathway.

We previously generated cytochrome P450 4F2 (CYP4F2) transgenic mice that have high levels of 20-hydroxyeicosatetraenoic acid (20-HETE) production; these mice exhibit both hypertension and hyperglycemia without insulin resistance. Currently, it is unclear whether and how 20-HETE affects insulin secretion, thus resulting in hyperglycemia. In this study, we found that 20-HETE attenuated glucose-stimulated insulin secretion (GSIS) in CYP4F2 transgenic mice as well as in rat insulinoma INS-1E cells treated with 0.5 µM 20-HETE. HET0016, a selective inhibitor of 20-HETE synthesis, reversed the reduction in GSIS leading to a decrease in blood glucose in the transgenic mice. Furthermore, the expression of glucose transporter 2 (Glut2), Ser473 phosphorylation of protein kinase B (AKT), and Ser9 phosphorylation of glycogen synthase kinase-3ß (GSK-3ß) were decreased in CYP4F2 transgenic mice compared with wild-type mice. In vitro experiments in INS-1E cells revealed that 20-HETE activated the AKT/GSK-3ß pathway and thereby decreased Glut2 expression by inhibiting activator protein 1 (AP-1). TWS119, a GSK-3ß selective inhibitor, blocked the 20-HETE-mediated reduction in Glut2 expression. Therefore, we concluded that 20-HETE inhibition of Glut2 contributes to the reduction in GSIS, at least in part, through the AKT/GSK-3ß/AP-1/Glut2 pathway.

Differences in IGF axis-related proteins in amniotic fluid of trisomy 21 and trisomy 18 using a multiple reaction monitoring approach.

OBJECTIVE: Trisomy 21 and trisomy 18 are the two most common chromosomal anomalies in live births. To find new biomarkers for aneuploidies and pathogenesis of fetal malformations, we measured insulin-like growth factor (IGF) axis-related proteins in amniotic fluid (AF) of pregnant women carrying trisomies 21 or 18 affected fetuses using multiple reaction monitoring (MRM) approach. METHOD: Eighty-five AF samples from pregnant women carrying either trisomy 21, trisomy 18, or normal fetuses were collected. IGF axis-related proteins in AF after serial treatments were quantitated with MRM method. The differential protein levels were also confirmed by western blot in AF without any treatment. RESULTS: The IGF type I receptor and pregnancy-associated plasma protein-A in AF of trisomy 21 (1.35 ± 0.32 and 13.36 ± 3.64 µg/mg protein) and trisomy 18 (1.39 ± 0.40 and 12.80 ± 1.84 µg/mg protein) were decreased versus normal controls (2.16 ± 0.59 and 23.77 ± 6.18 µg/mg protein). IGF binding protein 5 was reduced in trisomy 18 (1.47 ± 0.33 vs 2.36 ± 0.77 µg/mg protein). These alterations were confirmed by western blot. The other proteins showed no significant difference between the three groups. CONCLUSION: Our data suggested that MRM can provide a powerful platform for the identification of biomarkers in AF that have crucial developmental effects in the aneuploid fetus.

Synergistical effect of 20-HETE and high salt on NKCC2 protein and blood pressure via ubiquitin-proteasome pathway.

We previously generated a cytochrome P450 4F2 (CYP4F2) transgenic mouse model and demonstrated that overexpressed CYP4F2 and overproduced 20-HETE in the kidneys contribute to the increase of blood pressure in the CYP4F2 transgenic mice with normal salt intake. We currently expect to elucidate a potential mechanism of salt-related hypertension whereby diverse levels of 20-HETE interact with dietary salt on Na(+)-K(+)-2Cl(-) cotransporter, isoform 2 (NKCC2) in the kidneys of the transgenic and wild-type mice with high salt intake. High salt intake reduced about 85 % abundance of renal NKCC2 protein in the transgenic mice and about 24 % in the wild-type mice by Western blot. Furthermore, we first found that NKCC2 was ubiquitinated and interacted with Nedd4-2 by immunoprecipitation in the transgenic mice with high salt intake. In addition, inhibition of 20-HETE synthesis or proteasome activity reversed the reduction of NKCC2 expression induced by 20-HETE and high salt intake. These results suggest that 20-HETE and high salt intake synergistically decrease the expression of NKCC2 protein via Nedd4-2-mediated ubiquitin-proteasome pathway, and thereby modulate natriuresis and blood pressure. We propose that diverse levels of 20-HETE have diverse effects on blood pressure in different salt concentrations.

Association of SLC38A4 and system A with abnormal fetal birth weight.

In this study, we aimed to explore the correlation between solute carrier family 38 member 4 (SLC38A4) and system A activity in human placentas from pregnancies with abnormal fetal birth weight. We collected placentas from consenting women immediately after their full-term babies were born, with normal, low birth weight or macrosomia, and used real-time PCR and Western blot analysis to detect the levels of SLC38A4 mRNA and protein [also known as sodium-coupled neutral amino acid transport protein 4 (SNAT4)]. Isotope incorporation assay was applied to measure system A activity in the placentas. Compared to the normal birth weight (NBW) group, placentas from the fetal macrosomia (FM) group had significantly increased levels of SLC38A4 mRNA and SNAT4 (both were increased by almost 2-fold; P<0.05), while no significant changes were detected in the placentas from the low birth weight (LBW) group. In addition, system A activity in the placentas from the FM and LBW groups was significantly different from that in the NBW group (1.2±0.20, 0.6±0.14 vs. 1.0±0.18, P<0.05). The data suggest that SNAT4 and system A have a strong association with abnormal fetal birth weight and that they may play a crucial role in fetal growth and development.

20-HETE induces hyperglycemia through the cAMP/PKA-PhK-GP pathway.

We previously generated cytochrome P450 4F2 (CYP4F2) transgenic mice and showed high 20-hydroxyeicosatetraenoic acid (20-HETE) production, which resulted in an elevation of blood pressure. However, it was unclear whether 20-HETE affected glucose metabolism. We measured fasting plasma glucose, insulin, hepatic CYP4F2 expression, and 20-HETE production by hepatic microsomes, and hepatic 20-HETE levels in transgenic mice. We also assessed glycogen phosphorylase (GP) activity and the cAMP/protein kinase A (PKA)-phosphorylase kinase (PhK)-GP pathway, as well as expressions of insulin receptor substrate 1 and glucose transporters in vivo and in vitro. The transgenic mice had overexpressed hepatic CYP4F2, high hepatic 20-HETE and fasting plasma glucose levels but normal insulin level. The GP activity was increased and the cAMP/PKA-PhK-GP pathway was activated in the transgenic mice compared with wild-type mice. Moreover, these alterations were eliminated with the addition of N-hydroxy-N'-(4-butyl-2 methylphenyl) formamidine, which is a selective 20-HETE inhibitor. The results were further validated in Bel7402 cells. In addition, the transgenic mice had functional insulin signaling, and 20-HETE had no effect on insulin signaling in Bel7402 cells, excluding that the observed hyperglycemia in CYP4F2 transgenic mice resulted from insulin dysfunction, because the target tissues were sensitive to insulin. Our study suggested that 20-HETE can induce hyperglycemia, at least in part, through the cAMP/PKA-PhK-GP pathway but not through the insulin-signaling pathway.