Maternal glucose homeostasis is impaired in mouse models of gestational cholestasis.

Women with intrahepatic cholestasis of pregnancy (ICP), a disorder characterised by raised serum bile acids, are at increased risk of developing gestational diabetes mellitus and have impaired glucose tolerance whilst cholestatic. FXR and TGR5 are modulators of glucose metabolism, and FXR activity is reduced in normal pregnancy, and further in ICP. We aimed to investigate the role of raised serum bile acids, FXR and TGR5 in gestational glucose metabolism using mouse models. Cholic acid feeding resulted in reduced pancreatic ß-cell proliferation and increased apoptosis in pregnancy, without altering insulin sensitivity, suggesting that raised bile acids affect ß-cell mass but are insufficient to impair glucose tolerance. Conversely, pregnant Fxr-/- and Tgr5-/- mice are glucose intolerant and have reduced insulin secretion in response to glucose challenge, and Fxr-/- mice are also insulin resistant. Furthermore, fecal bile acids are reduced in pregnant Fxr-/- mice. Lithocholic acid and deoxycholic acid, the principal ligands for TGR5, are decreased in particular. Therefore, we propose that raised serum bile acids and reduced FXR and TGR5 activity contribute to the altered glucose metabolism observed in ICP.

Obeticholic acid ameliorates dyslipidemia but not glucose tolerance in mouse model of gestational diabetes.

Metabolism alters markedly with advancing gestation, characterized by progressive insulin resistance, dyslipidemia, and raised serum bile acids. The nuclear receptor farnesoid X receptor (FXR) has an integral role in bile acid homeostasis and modulates glucose and lipid metabolism. FXR is known to be functionally suppressed in pregnancy. The FXR agonist, obeticholic acid (OCA), improves insulin sensitivity in patients with type 2 diabetes with nonalcoholic fatty liver disease. We therefore hypothesized that OCA treatment during pregnancy could improve disease severity in a mouse model of gestational diabetes mellitus (GDM). C57BL/6J mice were fed a high-fat diet (HFD; 60% kcal from fat) for 4 wk before and throughout pregnancy to induce GDM. The impact of the diet supplemented with 0.03% OCA throughout pregnancy was studied. Pregnant HFD-fed mice displayed insulin resistance and dyslipidemia. OCA significantly reduced plasma cholesterol concentrations in nonpregnant and pregnant HFD-fed mice (by 22.4%, P < 0.05 and 36.4%, P < 0.001, respectively) and reduced the impact of pregnancy on insulin resistance but did not change glucose tolerance. In nonpregnant HFD-fed mice, OCA ameliorated weight gain, reduced mRNA expression of inflammatory markers in white adipose tissue, and reduced plasma glucagon-like peptide 1 concentrations (by 62.7%, P < 0.01). However, these effects were not evident in pregnant mice. OCA administration can normalize plasma cholesterol levels in a mouse model of GDM. However, the absence of several of the effects of OCA in pregnant mice indicates that the agonistic action of OCA is not sufficient to overcome many metabolic consequences of the pregnancy-associated reduction in FXR activity.

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.

Paternal cholestasis exacerbates obesity-associated hypertension in male offspring but is prevented by paternal ursodeoxycholic acid treatment.

BACKGROUND: Obesity is a heterogeneous phenotype and risk associations to non-communicable diseases such as cardiovascular disease and type 2 diabetes are influenced by several factors. The paternal metabolic status at the time of conception influences offspring susceptibility to developing obesity and adiposity-associated cardiometabolic disease. Cholestatic liver diseases are characterized by raised circulating serum bile acid levels and dyslipidemia, and are commonly treated with ursodeoxycholic acid (UDCA). We hypothesized that paternal cholestasis alters offspring susceptibility to developing obesity and adiposity-associated cardiometabolic disease and that this may be modified by paternal UDCA treatment. METHODS: Cholestasis was induced in male C57BL/6 mice with a 0.5% cholic acid (CA)-supplemented diet for 10 weeks prior to mating with normal chow (NC)-fed females. Offspring of cholestatic and NC-fed fathers were fed either a NC diet or challenged with an obesogenic 'western diet' (WD) from 12 weeks of age. Offspring body weight and cardiometabolic function were assessed, and the impact of treatment of paternal cholestasis with UDCA was evaluated. RESULTS: Male offspring (18 weeks old) of cholestatic fathers challenged with WD had raised fasting insulin, hepatic triglyceride content and serum cholesterol levels compared to diet-matched controls. At 25-29 weeks of age, WD-fed male offspring of cholestatic fathers had higher systolic and diastolic blood pressure than controls and this was prevented by paternal UDCA treatment. In contrast, WD-challenged female offspring of cholestatic fathers showed improved glucose tolerance compared to controls. CONCLUSIONS: We demonstrated in our model of paternal cholestasis that offspring susceptibility to adiposity-associated cardiometabolic disease is affected in a sex-specific manner and paternal UDCA treatment had a protective effect against hypertension in the obese male offspring. The most prevalent human cholestatic conditions are primary sclerosing cholangitis and primary biliary cholangitis. These findings are of clinical relevance to children of men with these conditions.

Bile acids and their respective conjugates elicit different responses in neonatal cardiomyocytes: role of Gi protein, muscarinic receptors and TGR5.

Bile acids are recognised as bioactive signalling molecules. While they are known to influence arrhythmia susceptibility in cholestasis, there is limited knowledge about the underlying mechanisms. To delineate mechanisms underlying fetal heart rhythm disturbances in cholestatic pregnancy, we used FRET microscopy to monitor cAMP release and contraction measurements in isolated rodent neonatal cardiomyocytes. The unconjugated bile acids CDCA, DCA and UDCA and, to a lesser extent, CA were found to be relatively potent agonists for the GPBAR1 (TGR5) receptor and elicit cAMP release, whereas all glyco- and tauro- conjugated bile acids are weak agonists. The bile acid-induced cAMP production does not lead to an increase in contraction rate, and seems to be mediated by the RI isoform of adenylate cyclase, unlike adrenaline-dependent release which is mediated by the RII isoform. In contrast, bile acids elicited slowing of neonatal cardiomyocyte contraction indicating that other signalling pathways are involved. The conjugated bile acids were found to be partial agonists of the muscarinic M2, but not sphingosin-1-phosphate-2, receptors, and act partially through the Gi pathway. Furthermore, the contraction slowing effect of unconjugated bile acids may also relate to cytotoxicity at higher concentrations.

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.

Gestational disruptions in metabolic rhythmicity of the liver, muscle, and placenta affect fetal size.

Maternal metabolic adaptations are essential for successful pregnancy outcomes. We investigated how metabolic gestational processes are coordinated, whether there is a functional link with internal clocks, and whether disruptions are related to metabolic abnormalities in pregnancy, by studying day/night metabolic pathways in murine models and samples from pregnant women with normally grown and large-for-gestational age infants. In early mouse pregnancy, expression of hepatic lipogenic genes was up-regulated and uncoupled from the hepatic clock. In late mouse pregnancy, rhythmicity of energy metabolism-related genes in the muscle followed the patterns of internal clock genes in this tissue, and coincided with enhanced lipid transporter expression in the fetoplacental unit. Diurnal triglyceride patterns were disrupted in human placentas from pregnancies with large-for-gestational age infants and this overlapped with an increase in BMAL1 expression. Metabolic adaptations in early pregnancy are uncoupled from the circadian clock, whereas in late pregnancy, energy availability is mediated by coordinated muscle-placenta metabolic adjustments linked to internal clocks. Placental triglyceride oscillations in the third trimester of human pregnancy are lost in large-for-gestational age infants and may be regulated by BMAL1. In summary, disruptions in metabolic and circadian rhythmicity are associated with increased fetal size, with implications for the pathogenesis of macrosomia.-Papacleovoulou, G., Nikolova, V., Oduwole, O., Chambers, J., Vazquez-Lopez, M., Jansen, E., Nicolaides, K., Parker, M., Williamson, C. Gestational disruptions in metabolic rhythmicity of the liver, muscle, and placenta affect fetal size.

Maternal cholestasis during pregnancy programs metabolic disease in offspring.

The intrauterine environment is a major contributor to increased rates of metabolic disease in adults. Intrahepatic cholestasis of pregnancy (ICP) is a liver disease of pregnancy that affects 0.5%-2% of pregnant women and is characterized by increased bile acid levels in the maternal serum. The influence of ICP on the metabolic health of offspring is unknown. We analyzed the Northern Finland birth cohort 1985-1986 database and found that 16-year-old children of mothers with ICP had altered lipid profiles. Males had increased BMI, and females exhibited increased waist and hip girth compared with the offspring of uncomplicated pregnancies. We further investigated the effect of maternal cholestasis on the metabolism of adult offspring in the mouse. Females from cholestatic mothers developed a severe obese, diabetic phenotype with hepatosteatosis following a Western diet, whereas matched mice not exposed to cholestasis in utero did not. Female littermates were susceptible to metabolic disease before dietary challenge. Human and mouse studies showed an accumulation of lipids in the fetoplacental unit and increased transplacental cholesterol transport in cholestatic pregnancy. We believe this is the first report showing that cholestatic pregnancy in the absence of altered maternal BMI or diabetes can program metabolic disease in the offspring.

Intrahepatic cholestasis of pregnancy levels of sulfated progesterone metabolites inhibit farnesoid X receptor resulting in a cholestatic phenotype.

UNLABELLED: Intrahepatic cholestasis of pregnancy (ICP) is the most prevalent pregnancy-specific liver disease and is associated with an increased risk of adverse fetal outcomes, including preterm labor and intrauterine death. The endocrine signals that cause cholestasis are not known but 3α-sulfated progesterone metabolites have been shown to be elevated in ICP, leading us to study the impact of sulfated progesterone metabolites on farnesoid X receptor (FXR)-mediated bile acid homeostasis pathways. Here we report that the 3ß-sulfated progesterone metabolite epiallopregnanolone sulfate is supraphysiologically raised in the serum of ICP patients. Mice challenged with cholic acid developed hypercholanemia and a hepatic gene expression profile indicative of FXR activation. However, coadministration of epiallopregnanolone sulfate with cholic acid exacerbated the hypercholanemia and resulted in aberrant gene expression profiles for hepatic bile acid-responsive genes consistent with cholestasis. We demonstrate that levels of epiallopregnanolone sulfate found in ICP can function as a partial agonist for FXR, resulting in the aberrant expression of bile acid homeostasis genes in hepatoma cell lines and primary human hepatocytes. Furthermore, epiallopregnanolone sulfate inhibition of FXR results in reduced FXR-mediated bile acid efflux and secreted FGF19. Using cofactor recruitment assays, we show that epiallopregnanolone sulfate competitively inhibits bile acid-mediated recruitment of cofactor motifs to the FXR-ligand binding domain. CONCLUSION: Our results reveal a novel molecular interaction between ICP-associated levels of the 3ß-sulfated progesterone metabolite epiallopregnanolone sulfate and FXR that couples the endocrine component of pregnancy in ICP to abnormal bile acid homeostasis.