Prognostic and mechanistic potential of progesterone sulfates in intrahepatic cholestasis of pregnancy and pruritus gravidarum

A challenge in obstetrics is to distinguish pathological symptoms from those associated with normal changes of pregnancy, typified by the need to differentiate whether gestational pruritus of the skin is an early symptom of intrahepatic cholestasis of pregnancy (ICP) or due to benign pruritus gravidarum. ICP is characterized by raised serum bile acids and complicated by spontaneous preterm labor and stillbirth. A biomarker for ICP would be invaluable for early diagnosis and treatment and to enable its differentiation from other maternal diseases. Three progesterone sulfate compounds, whose concentrations have not previously been studied, were newly synthesized and assayed in the serum of three groups of ICP patients and found to be significantly higher in ICP at 9-15 weeks of gestation and prior to symptom onset (group 1 cases/samples: ICP n = 35/80, uncomplicated pregnancy = 29/100), demonstrating that all three progesterone sulfates are prognostic for ICP. Concentrations of progesterone sulfates were associated with itch severity and, in combination with autotaxin, distinguished pregnant women with itch that would subsequently develop ICP from pruritus gravidarum (group 2: ICP n = 41, pruritus gravidarum n = 14). In a third group of first-trimester samples all progesterone sulfates were significantly elevated in serum from low-risk asymptomatic women who subsequently developed ICP (ICP/uncomplicated pregnancy n = 54/51). Finally, we show mechanistically that progesterone sulfates mediate itch by evoking a Tgr5-dependent scratch response in mice. Conclusion: Our discovery that sulfated progesterone metabolites are a prognostic indicator for ICP will help predict onset of ICP and distinguish it from benign pruritus gravidarum, enabling targeted obstetric care to a high-risk population. Delineation of a progesterone sulfate-TGR5 pruritus axis identifies a therapeutic target for itch management in IC

A Phosphoinositide 3-Kinase/Phospholipase Cgamma1 Pathway Regulates Fibroblast Growth Factor-Induced Capillary Tube Formation

Background: The fibroblast growth factors (FGFs) are key regulators of embryonic development, tissue homeostasis and tumour angiogenesis. Binding of FGFs to their receptor(s) results in activation of several intracellular signalling cascades including phosphoinositide 3-kinase (PI3K) and phospholipase C (PLC)gamma 1. Here we investigated the basic FGF (FGF-2)-mediated activation of these enzymes in human umbilical vein endothelial cells (HUVECs) and defined their role in FGF-2-dependent cellular functions.Methodology/Principal Findings: We show that FGF-2 activates PLC gamma 1 in HUVECs measured by analysis of total inositol phosphates production upon metabolic labelling of cells and intracellular calcium increase. We further demonstrate that FGF-2 activates PI3K, assessed by analysing accumulation of its lipid product phosphatidylinositol-3,4,5-P-3 using TLC and confocal microscopy analysis. PI3K activity is required for FGF-2-induced PLC gamma 1 activation and the PI3K/PLC gamma 1 pathway is involved in FGF-2-dependent cell migration, determined using Transwell assay, and in FGF-2-induced capillary tube formation (tubulogenesis assays in vitro). Finally we show that PI3K-dependent PLC gamma 1 activation regulates FGF-2-mediated phosphorylation of Akt at its residue Ser473, determined by Western blotting analysis. This occurs through protein kinase C (PKC)alpha activation since dowregulation of PKC alpha expression using specific siRNA or blockade of its activity using chemical inhibition affects the FGF-2-dependent Ser473 Akt phosphorylation. Furthermore inhibition of PKC alpha blocks FGF-2-dependent cell migration.Conclusion/Significance: These data elucidate the role of PLC gamma 1 in FGF-2 signalling in HUVECs demonstrating its key role in FGF-2-dependent tubulogenesis. Furthermore these data unveil a novel role for PLC gamma 1 as a mediator of PI3K-dependent Akt activation and as a novel key regulator of different Akt-dependent processes

Changes in LXR signaling influence early-pregnancy lipogenesis and protect against dysregulated fetoplacental lipid homeostasis

Human pregnancy is associated with enhanced de novo lipogenesis in the early stages followed by hyperlipidemia during advanced gestation. Liver X receptors (LXRs) are oxysterol-activated nuclear receptors that stimulate de novo lipogenesis and also promote the efflux of cholesterol from extrahepatic tissues followed by its transport back to the liver for biliary excretion. Although LXR is recognized as a master regulator of triglyceride and cholesterol homeostasis, it is unknown whether it facilitates the gestational adaptations in lipid metabolism. To address this question, biochemical profiling, protein quantification, and gene expression studies were used, and gestational metabolic changes in T0901317-treated wild-type mice and Lxrab-/- mutants were investigated. Here, we show that altered LXR signaling contributes to the enhanced lipogenesis in early pregnancy by increasing the expression of hepatic Fas and stearoyl-CoA desaturase 1 (Scd1). Both the pharmacological activation of LXR with T0901317 and the genetic ablation of its two isoforms disrupted the increase in hepatic fatty acid biosynthesis and the development of hypertriglyceridemia during early gestation. We also demonstrate that absence of LXR enhances maternal white adipose tissue lipolysis, causing abnormal accumulation of triglycerides, cholesterol, and free fatty acids in the fetal liver. Together, these data identify LXR as an important factor in early-pregnancy lipogenesis that is also necessary to protect against abnormalities in fetoplacental lipid homeostasis

LKB1 is required for hepatic bile acid transport and canalicular membrane integrity in mice

LKB1 is a ‘master’ protein kinase implicated in the regulation of metabolism, cell proliferation, cell polarity and tumorigenesis. However, the long-term role of LKB1 in hepatic function is unknown. In the present study, it is shown that hepatic LKB1 plays a key role in liver cellular architecture and metabolism. We report that liver-specific deletion of LKB1 in mice leads to defective canaliculi and bile duct formation, causing impaired bile acid clearance and subsequent accumulation of bile acids in serum and liver. Concomitant with this, it was found that the majority of BSEP (bile salt export pump) was retained in intracellular pools rather than localized to the canalicular membrane in hepatocytes from LLKB1KO (liver-specific Lkb1-knockout) mice. Together, these changes resulted in toxic accumulation of bile salts, reduced liver function and failure to thrive. Additionally, circulating LDL (low-density lipoprotein)-cholesterol and non-esterified cholesterol levels were increased in LLKB1KO mice with an associated alteration in red blood cell morphology and development of hyperbilirubinaemia. These results indicate that LKB1 plays a critical role in bile acid homoeostasis and that lack of LKB1 in the liver results in cholestasis. These findings indicate a novel key role for LKB1 in the development of hepatic morphology and membrane targeting of canalicular proteins

Refugee crisis, imperialism and pitiless wars on the poor

According to the UN Refugee Agency, 59.5 million people around the world were forcibly displaced in 2014. The numbers are particularly high in countries which have been subject to a process of ‘redrawing the map’ by imperial powers or their regional allies. The response to the recent developments – a stage which has been dubbed as ‘refugee crisis’ – is as polarising and as problematic as before. On the one hand we have witnessed the heroic acts not only of the refugees themselves who moved collectively and refused to queue ‘orderly’ in the immigration lines, but also the magnificent response of citizens in all over Europe who rushed to feed, clothe, accommodate and welcome them. In contrast the overwhelming institutional response by ‘liberal’ states has been, and remains, depressingly illiberal. The official response to this humanitarian crisis – which is after all the product of ‘humanitarian interventions’ – has nothing to do with whether or not Europe can cope with a ‘swarm of people’ aiming to exploit the ‘host’ countries. It is to do with managing a massive reserve army of labour. Forced migration is not only a product of this staggering inequality but also an important element of how that inequality is produced, maintained and managed

Enhanced microbial bile acid deconjugation and impaired ileal uptake in pregnancy repress intestinal regulation of bile acid synthesis

Pregnancy is associated with progressive hypercholanemia, hypercholesterolemia, and hypertriglyceridemia, which can result in metabolic disease in susceptible women. Gut signals modify hepatic homeostatic pathways, linking intestinal content to metabolic activity. We sought to identify whether enteric endocrine signals contribute to raised serum bile acids observed in human and murine pregnancies, by measuring fibroblast growth factor (FGF) 19/15 protein and mRNA levels, and 7α-hydroxy-4-cholesten-3-one. Terminal ileal farnesoid X receptor (FXR)-mediated gene expression and apical sodium bile acid transporter (ASBT) protein concentration were measured by qPCR and western blotting. Shotgun whole-genome sequencing and ultra-performance liquid chromatography tandem mass spectrometry were used to determine the cecal microbiome and metabonome. Targeted and untargeted pathway analyses were performed to predict the systemic effects of the altered metagenome and metabolite profiles. Dietary CA supplementation was used to determine whether the observed alterations could be overcome by intestinal bile acids functioning as FXR agonists. Human and murine pregnancy were associated with reduced intestinal FXR signaling, with lower FGF19/15 and resultant increased hepatic bile acid synthesis. Terminal ileal ASBT protein was reduced in murine pregnancy. Cecal bile acid conjugation was reduced in pregnancy because of elevated bile salt hydrolase-producing Bacteroidetes. CA supplementation induced intestinal FXR signaling, which was not abrogated by pregnancy, with strikingly similar changes to the microbiota and metabonome as identified in pregnancy. Conclusion: The altered intestinal microbiota of pregnancy enhance bile acid deconjugation, reducing ileal bile acid uptake and lowering FXR induction in enterocytes. This exacerbates the effects mediated by reduced bile acid uptake transporters in pregnancy. Thus, in pregnant women and mice, there is reduced FGF19/15-mediated hepatic repression of hepatic bile acid synthesis, resulting in hypercholanemia

Prognostic and Mechanistic Potential of Progesterone Sulfates in Intrahepatic Cholestasis of Pregnancy and Pruritus Gravidarum

A challenge in obstetrics is to distinguish pathological symptoms from those associated with normal changes of pregnancy, typified by the need to differentiate whether gestational pruritus of the skin is an early symptom of intrahepatic cholestasis of pregnancy (ICP) or due to benign pruritus gravidarum. ICP is characterized by raised serum bile acids and complicated by spontaneous preterm labor and stillbirth. A biomarker for ICP would be invaluable for early diagnosis and treatment and to enable its differentiation from other maternal diseases. Three progesterone sulfate compounds, whose concentrations have not previously been studied, were newly synthesized and assayed in the serum of three groups of ICP patients and found to be significantly higher in ICP at 9-15 weeks of gestation and prior to symptom onset (group 1 cases/samples: ICP n = 35/80, uncomplicated pregnancy = 29/100), demonstrating that all three progesterone sulfates are prognostic for ICP. Concentrations of progesterone sulfates were associated with itch severity and, in combination with autotaxin, distinguished pregnant women with itch that would subsequently develop ICP from pruritus gravidarum (group 2: ICP n = 41, pruritus gravidarum n = 14). In a third group of first-trimester samples all progesterone sulfates were significantly elevated in serum from low-risk asymptomatic women who subsequently developed ICP (ICP/uncomplicated pregnancy n = 54/51). Finally, we show mechanistically that progesterone sulfates mediate itch by evoking a Tgr5-dependent scratch response in mice. Conclusion: Our discovery that sulfated progesterone metabolites are a prognostic indicator for ICP will help predict onset of ICP and distinguish it from benign pruritus gravidarum, enabling targeted obstetric care to a high-risk population. Delineation of a progesterone sulfate-TGR5 pruritus axis identifies a therapeutic target for itch management in ICP

A comprehensive analysis of common genetic variation around six candidate loci for intrahepatic cholestasis of pregnancy.

OBJECTIVES: Intrahepatic cholestasis of pregnancy (ICP) has a complex etiology with a significant genetic component. Heterozygous mutations of canalicular transporters occur in a subset of ICP cases and a population susceptibility allele (p.444A) has been identified in ABCB11. We sought to expand our knowledge of the detailed genetic contribution to ICP by investigation of common variation around candidate loci with biological plausibility for a role in ICP (ABCB4, ABCB11, ABCC2, ATP8B1, NR1H4, and FGF19). METHODS: ICP patients (n=563) of white western European origin and controls (n=642) were analyzed in a case-control design. Single-nucleotide polymorphism (SNP) markers (n=83) were selected from the HapMap data set (Tagger, Haploview 4.1 (build 22)). Genotyping was performed by allelic discrimination assay on a robotic platform. Following quality control, SNP data were analyzed by Armitage's trend test. RESULTS: Cochran-Armitage trend testing identified six SNPs in ABCB11 together with six SNPs in ABCB4 that showed significant evidence of association. The minimum Bonferroni corrected P value for trend testing ABCB11 was 5.81×10(-4) (rs3815676) and for ABCB4 it was 4.6×10(-7)(rs2109505). Conditional analysis of the two clusters of association signals suggested a single signal in ABCB4 but evidence for two independent signals in ABCB11. To confirm these findings, a second study was performed in a further 227 cases, which confirmed and strengthened the original findings. CONCLUSIONS: Our analysis of a large cohort of ICP cases has identified a key role for common variation around the ABCB4 and ABCB11 loci, identified the core associations, and expanded our knowledge of ICP susceptibility

Impact of gastrointestinal tract variability on oral drug absorption and pharmacokinetics : an UNGAP review

The absorption of oral drugs is frequently plagued by significant variability with potentially serious therapeutic consequences. The source of variability can be traced back to interindividual variability in physiology, differences in special populations (age- and disease-dependent), drug and formulation properties, or food-drug interactions. Clinical evidence for the impact of some of these factors on drug pharmacokinetic variability is mounting: e.g. gastric pH and emptying time, small intestinal fluid properties, differences in pediatrics and the elderly, and surgical changes in gastrointestinal anatomy. However, the link of colonic factors variability (transit time, fluid composition, microbiome), sex differences (male vs. female) and gut-related diseases (chronic constipation, anorexia and cachexia) to drug absorption variability has not been firmly established yet. At the same time, a way to decrease oral drug pharmacokinetic variability is provided by the pharmaceutical industry: clinical evidence suggests that formulation approaches employed during drug development can decrease the variability in oral exposure. This review outlines the main drivers of oral drug exposure variability and potential approaches to overcome them, while highlighting existing knowledge gaps and guiding future studies in this area