Reversal of insulin resistance in people with obesity by lifestyle-induced weight loss does not impact the proportion of circulating 12α-hydroxylated bile acids.

AIM: Bile acids (BAs) are implicated in the pathogenesis of several metabolic syndrome-related diseases, including insulin resistance (IR) and type 2 diabetes (T2D). It has been reported that IR and T2D are associated with an increased ratio of 12α/non-12α-hydroxylated BAs in the circulating BA pool. It is, however, unknown whether the improvement of insulin sensitivity inversely affects BA composition in humans. Therefore, we assessed whether lifestyle-induced weight loss induces changes in BA metabolism in people with obesity, with or without T2D, and if these changes are associated with metabolic parameters. MATERIALS AND METHODS: Individual BAs and C4 were quantified by ultra-high-performance liquid chromatography-tandem mass spectrometry in plasma samples collected from two cohorts of people with obesity (OB) and with T2D and obesity (T2D), before and after a lifestyle intervention. RESULTS: Lifestyle-induced weight loss improved glycaemic control in both cohorts, with plasma BA concentrations not affected by the lifestyle interventions. The ratio of 12α/non-12α-hydroxylated BAs remained unchanged in OB (p = .178) and even slightly increased upon intervention in T2D (p = .0147). Plasma C4 levels were unaffected in OB participants (p = .20) but significantly reduced in T2D after intervention (p = .0003). There were no significant correlations between the ratio of 12α/non-12α-hydroxylated BAs and glucose, insulin, or homeostatic model assessment-IR, nor in plasma triglycerides, low-density lipoprotein cholesterol, lipoprotein (a) in the T2D cohort. CONCLUSIONS: Lifestyle-induced weight loss did improve glycaemic control but did not affect BA concentrations. Improvements in insulin sensitivity were not associated with changes in BA parameters in people with obesity, with or without T2D.

Hyperglycaemia, pregnancy outcomes and maternal metabolic disease risk during pregnancy and lactation in a lean gestational diabetes mouse model.

Hyperglycaemia in pregnancy (HIP) is a pregnancy complication characterized by mild to moderate hyperglycaemia that negatively impacts short- and long-term health of mother and child. However, relationships between severity and timing of pregnancy hyperglycaemia and postpartum outcomes have not been systemically investigated. We investigated the impact of hyperglycaemia developing during pregnancy (gestational diabetes mellitus, GDM) or already present pre-mating (pre-gestational diabetes mellitus, PDM) on maternal health and pregnancy outcomes. GDM and PDM were induced in C57BL/6NTac mice by combined 60% high fat diet (HF) and low dose streptozotocin (STZ). Animals were screened for PDM prior to mating, and all underwent an oral glucose tolerance test on gestational day (GD)15. Tissues were collected at GD18 or at postnatal day (PN)15. Among HFSTZ-treated dams, 34% developed PDM and 66% developed GDM, characterized by impaired glucose-induced insulin release and inadequate suppression of endogenous glucose production. No increased adiposity or overt insulin resistance was observed. Furthermore, markers of non-alcoholic fatty liver disease (NAFLD) were significantly increased in PDM at GD18 and were positively correlated with basal glucose levels at GD18 in GDM dams. By PN15, NAFLD markers were also increased in GDM dams. Only PDM affected pregnancy outcomes such as litter size. Our findings indicate that GDM and PDM, resulting in disturbances of maternal glucose homeostasis, increase the risk of postpartum NAFLD development, related to the onset and severity of pregnancy hyperglycaemia. These findings signal a need for earlier monitoring of maternal glycaemia and more rigorous follow-up of maternal health after GDM and PDM pregnancy in humans. KEY POINTS: We studied the impact of high-fat diet/streptozotocin induced hyperglycaemia in pregnancy in mice and found that this impaired glucose tolerance and insulin release. Litter size and embryo survival were compromised by pre-gestational, but not by gestational, diabetes. Despite postpartum recovery from hyperglycaemia in a majority of dams, liver disease markers were further elevated by postnatal day 15. Maternal liver disease markers were associated with the severity of hyperglycaemia at gestational day 18. The association between hyperglycaemic exposure and non-alcoholic fatty liver disease signals a need for more rigorous monitoring and follow-up of maternal glycaemia and health in diabetic pregnancy in humans.

Gut microbiota depletion aggravates bile acid-induced liver pathology in mice with a human-like bile acid composition.

Cyp2c70-deficient mice have a human-like bile acid (BA) composition due to their inability to convert chenodeoxycholic acid (CDCA) into rodent-specific muricholic acids (MCAs). However, the hydrophobic BA composition in these animals is associated with liver pathology. Although Cyp2c70-ablation has been shown to alter gut microbiome composition, the impact of gut bacteria on liver pathology in Cyp2c70-/- mice remains to be established. Therefore, we treated young-adult male and female wild-type (WT) and Cyp2c70-/- mice with antibiotics (AB) with broad specificity to deplete the gut microbiota and assessed the consequences on BA metabolism and liver pathology. Female Cyp2c70-/- mice did not tolerate AB treatment, necessitating premature termination of the experiment. Male Cyp2c70-/- mice did tolerate AB but showed markedly augmented liver pathology after 6 weeks of treatment. Dramatic downregulation of hepatic Cyp8b1 expression (-99%) caused a reduction in the proportions of 12α-hydroxylated BAs in the circulating BA pools of AB-treated male Cyp2c70-/- mice. Interestingly, the resulting increased BA hydrophobicity strongly correlated with various indicators of liver pathology. Moreover, genetic inactivation of Cyp8b1 in livers of male Cyp2c70-/- mice increased liver pathology, while addition of ursodeoxycholic acid to the diet prevented weight loss and liver pathology in AB-treated female Cyp2c70-/- mice. In conclusion, depletion of gut microbiota in Cyp2c70-/- mice aggravates liver pathology at least in part by increasing the hydrophobicity of the circulating BA pool. These findings highlight that the potential implications of AB administration to cholestatic patients should be evaluated in a systematic manner.

Perinatal exposure to UDCA prevents neonatal cholestasis in Cyp2c70-/- mice with human-like bile acids.

BACKGROUND: Cyp2c70-/- mice with a human-like bile acid (BA) composition display features of neonatal cholestasis. We assessed whether perinatal ursodeoxycholic acid (UDCA) exposure prevents neonatal cholestasis in Cyp2c70-/- mice and reduces cholangiopathy development later in life. METHODS: Cyp2c70+/- males were crossed with Cyp2c70+/- females fed either a regular chow diet or a 0.1% UDCA-containing diet during breeding, gestation, and suckling. Cholestasis and liver function parameters were assessed in their Cyp2c70-/- and wild-type offspring at 3 and 8 weeks of age. RESULTS: Three-week-old Cyp2c70-/- pups showed features of neonatal cholestasis, including elevated plasma BAs and transaminases, which were completely prevented in Cyp2c70-/- pups upon perinatal UDCA exposure. In addition, UDCA administration to the dams corrected altered hepatic gene expression patterns in Cyp2c70-/- pups, reduced markers of fibrogenesis and inflammation, and prevented cholangiocyte proliferation. Yet, these beneficial effects of perinatal UDCA exposure were not retained into adulthood upon discontinuation of treatment. CONCLUSION: Perinatal exposure of Cyp2c70-/- mice to UDCA has beneficial effects on liver function parameters, supporting a direct role of BA hydrophobicity in the development of neonatal cholestasis in these mice. However, prevention of neonatal cholestasis in Cyp2c70-/- mice has no long-lasting effects on liver pathophysiology. IMPACT: This is the first study showing that perinatal UDCA exposure prevents features of neonatal cholestasis that are observed in mice with a human-like bile acid composition, i.e., Cyp2c70-/- mice. Perinatal UDCA exposure of Cyp2c70-/- pups leads to UDCA enrichment in their circulating bile acid pool and, consequently, to a reduced hydrophobicity of biliary bile acids. Perinatal UDCA exposure of Cyp2c70-/- pups has no long-lasting effects on the development of cholangiopathy after discontinuation of treatment. The results in this study expand current knowledge regarding acute and long-lasting effects of UDCA treatment in early life.

Intestinal Farnesoid X Receptor Modulates Duodenal Surface Area but Does Not Control Glucose Absorption in Mice.

Bile acids facilitate the intestinal absorption of dietary lipids and act as signalling molecules in the maintenance of metabolic homeostasis. Farnesoid X receptor (FXR) is a bile acid-responsive nuclear receptor involved in bile acid metabolism, as well as lipid and glucose homeostasis. Several studies have suggested a role of FXR in the control of genes regulating intestinal glucose handling. We applied a novel dual-label glucose kinetic approach in intestine-specific FXR-/- mice (iFXR-KO) to directly assess the role of intestinal FXR in glucose absorption. Although iFXR-KO mice showed decreased duodenal expression of hexokinase 1 (Hk1) under obesogenic conditions, the assessment of glucose fluxes in these mice did not show a role for intestinal FXR in glucose absorption. FXR activation with the specific agonist GS3972 induced Hk1, yet the glucose absorption rate remained unaffected. FXR activation increased the duodenal villus length in mice treated with GS3972, while stem cell proliferation remained unaffected. Accordingly, iFXR-KO mice on either chow, short or long-term HFD feeding displayed a shorter villus length in the duodenum compared to wild-type mice. These findings indicate that delayed glucose absorption reported in whole-body FXR-/- mice is not due to the absence of intestinal FXR. Yet, intestinal FXR does have a role in the small intestinal surface area.

Low production of 12α-hydroxylated bile acids prevents hepatic steatosis in Cyp2c70-/- mice by reducing fat absorption.

Bile acids (BAs) play important roles in lipid homeostasis, and BA signaling pathways serve as therapeutic targets for nonalcoholic fatty liver disease (NAFLD). Recently, we generated cytochrome P450, family 2, subfamily C, polypeptide 70 (Cyp2c70-/-) mice with a human-like BA composition lacking mouse-/rat-specific muricholic acids to accelerate translation from mice to humans. We employed this model to assess the consequences of a human-like BA pool on diet-induced obesity and NAFLD development. Male and female Cyp2c70-/- mice and WT littermates were challenged with a 12-week high-fat Western-type diet (WTD) supplemented with 0.25% cholesterol. Cyp2c70 deficiency induced a hydrophobic BA pool with high abundances of chenodeoxycholic acid, particularly in females, because of sex-dependent suppression of sterol 12α-hydroxylase (Cyp8b1). Plasma transaminases were elevated, and hepatic fibrosis was present in Cyp2c70-/- mice, especially in females. Surprisingly, female Cyp2c70-/- mice were resistant to WTD-induced obesity and hepatic steatosis, whereas male Cyp2c70-/- mice showed similar adiposity and moderately reduced steatosis compared with WT controls. Both intestinal cholesterol and FA absorption were reduced in Cyp2c70-/- mice, the latter more strongly in females, despite unaffected biliary BA secretion rates. Intriguingly, the biliary ratio 12α-/non-12α-hydroxylated BAs significantly correlated with FA absorption and hepatic triglyceride content as well as with specific changes in gut microbiome composition. The hydrophobic human-like BA pool in Cyp2c70-/- mice prevents WTD-induced obesity in female mice and NAFLD development in both genders, primarily because of impaired intestinal fat absorption. Our data point to a key role for 12α-hydroxylated BAs in control of intestinal fat absorption and modulation of gut microbiome composition.

Altered bile acid kinetics contribute to postprandial hypoglycaemia after Roux-en-Y gastric bypass surgery.

BACKGROUND/OBJECTIVES: Bile acids (BA) act as detergents in intestinal fat absorption and as modulators of metabolic processes via activation of receptors such as FXR and TGR5. Elevated plasma BA as well as increased intestinal BA signalling to promote GLP-1 release have been implicated in beneficial health effects of Roux-en-Y gastric bypass surgery (RYGB). Whether BA also contribute to the postprandial hypoglycaemia that is frequently observed post-RYGB is unknown. METHODS: Plasma BA, fibroblast growth factor 19 (FGF19), 7α-hydroxy-4-cholesten-3-one (C4), GLP-1, insulin and glucose levels were determined during 3.5 h mixed-meal tolerance tests (MMTT) in subjects after RYGB, either with (RYGB, n = 11) or without a functioning gallbladder due to cholecystectomy (RYGB-CC, n = 11). Basal values were compared to those of age, BMI and sex-matched obese controls without RYGB (n = 22). RESULTS: Fasting BA as well as FGF19 levels were elevated in RYGB and RYGB-CC subjects compared to non-bariatric controls, without significant differences between RYGB and RYGB-CC. Postprandial hypoglycaemia was observed in 8/11 RYGB-CC and only in 3/11 RYGB. Subjects who developed hypoglycaemia showed higher postprandial BA levels coinciding with augmented GLP-1 and insulin responses during the MMTT. The nadir of plasma glucose concentrations after meals showed a negative relationship with postprandial BA peaks. Plasma C4 was lower during MMTT in subjects experiencing hypoglycaemia, indicating lower hepatic BA synthesis. Computer simulations revealed that altered intestinal transit underlies the occurrence of exaggerated postprandial BA responses in hypoglycaemic subjects. CONCLUSION: Altered BA kinetics upon ingestion of a meal, as frequently observed in RYGB-CC subjects, appear to contribute to postprandial hypoglycaemia by stimulating intestinal GLP-1 release.

An early-life diet containing large phospholipid-coated lipid globules programmes later-life postabsorptive lipid trafficking in high-fat diet- but not in low-fat diet-fed mice.

Feeding mice in early life a diet containing an experimental infant milk formula (Nuturis®; eIMF), with a lipid structure similar to human milk, transiently lowered body weight (BW) and fat mass gain upon Western-style diet later in life, when compared with mice fed diets based on control IMF (cIMF). We tested the hypothesis that early-life eIMF feeding alters the absorption or the postabsorptive trafficking of dietary lipids in later life. Male C57BL/6JOlaHsd mice were fed eIMF/cIMF from postnatal day 16-42, followed by low- (LFD, American Institute of Nutrition (AIN)-93 G, 7 wt% fat) or high-fat diet (HFD, D12451, 24 wt% fat) until day 63-70. Lipid absorption rate and tissue concentrations were determined after intragastric administration of stable isotope (2H or 13C) labelled lipids in separate groups. Lipid enrichments in plasma and tissues were analysed using GC-MS. The rate of triolein absorption was similar between eIMF and cIMF fed LFD: 3·2 (sd 1·8) and 3·9 (sd 2·1) and HFD: 2·6 (sd 1·7) and 3·8 (sd 3·0) % dose/ml per h. Postabsorptive lipid trafficking, that is, concentrations of absorbed lipids in tissues, was similar in the eIMF and cIMF groups after LFD. Tissue levels of absorbed TAG after HFD feeding were lower in heart (-42 %) and liver (-46 %), and higher in muscle (+81 %, all P < 0·05) in eIMF-fed mice. In conclusion, early-life IMF diet affected postabsorptive trafficking of absorbed lipids after HFD, but not LFD. Changes in postabsorptive lipid trafficking could underlie the observed lower BW and body fat accumulation in later life upon a persistent long-term obesogenic challenge.

A human-like bile acid pool induced by deletion of hepatic Cyp2c70 modulates effects of FXR activation in mice.

Bile acids (BAs) facilitate intestinal absorption of lipid-soluble nutrients and modulate various metabolic pathways through the farnesoid X receptor (FXR) and Takeda G-protein-coupled receptor 5. These receptors are targets for therapy in cholestatic and metabolic diseases. However, dissimilarities in BA metabolism between humans and mice complicate translation of preclinical data. Cytochrome P450 family 2 subfamily c polypeptide 70 (CYP2C70) was recently proposed to catalyze the formation of rodent-specific muricholic acids (MCAs). With CRISPR/Cas9-mediated somatic genome editing, we generated an acute hepatic Cyp2c70 knockout mouse model (Cyp2c70ako) to clarify the role of CYP2C70 in BA metabolism in vivo and evaluate whether its activity modulates effects of pharmacologic FXR activation on cholesterol homeostasis. In Cyp2c70ako mice, chenodeoxycholic acid (CDCA) increased at the expense of ßMCA, resulting in a more hydrophobic human-like BA pool. Tracer studies demonstrated that, in vivo, CYP2C70 catalyzes the formation of ßMCA primarily by sequential 6ß-hydroxylation and C7-epimerization of CDCA, generating αMCA as an intermediate metabolite. Physiologically, the humanized BA composition in Cyp2c70ako mice blunted the stimulation of fecal cholesterol disposal in response to FXR activation compared with WT mice, predominantly due to reduced stimulation of transintestinal cholesterol excretion. Thus, deletion of hepatic Cyp2c70 in adult mice translates into a human-like BA pool composition and impacts the response to pharmacologic FXR activation. This Cyp2c70ako mouse model may be a useful tool for future studies of BA signaling and metabolism that informs human disease development and treatment.

Transintestinal and Biliary Cholesterol Secretion Both Contribute to Macrophage Reverse Cholesterol Transport in Rats-Brief Report.

OBJECTIVE: Reverse cholesterol transport comprises efflux of cholesterol from macrophages and its subsequent removal from the body with the feces and thereby protects against formation of atherosclerotic plaques. Because of lack of suitable animal models that allow for evaluation of the respective contributions of biliary cholesterol secretion and transintestinal cholesterol excretion (TICE) to macrophage reverse cholesterol transport under physiological conditions, the relative importance of both pathways in this process has remained controversial. APPROACH AND RESULTS: To separate cholesterol traffic via the biliary route from TICE, bile flow was mutually diverted between rats, continuously, for 3 days. Groups of 2 weight-matched rats were designated as a pair, and both rats were equipped with cannulas in the bile duct and duodenum. Bile from rat 1 was diverted to the duodenum of rat 2, whereas bile from rat 2 was rerouted to the duodenum of rat 1. Next, rat 1 was injected with [3H]cholesterol-loaded macrophages. [3H]Cholesterol secreted via the biliary route was consequently diverted to rat 2 and could thus be quantified from the feces of that rat. On the other hand, [3H]cholesterol tracer in the feces of rat 1 reflected macrophage-derived cholesterol excreted via TICE. Using this setup, we found that 63% of the label secreted with the fecal neutral sterols had travelled via the biliary route, whereas 37% was excreted via TICE. CONCLUSIONS: TICE and biliary cholesterol secretion contribute to macrophage reverse cholesterol transport in rats. The majority of macrophage-derived cholesterol is however excreted via the hepatobiliary route.

Difference in postprandial GLP-1 response despite similar glucose kinetics after consumption of wheat breads with different particle size in healthy men.

PURPOSE: Underlying mechanisms of the beneficial health effects of low glycemic index starchy foods are not fully elucidated yet. We varied the wheat particle size to obtain fiber-rich breads with a high and low glycemic response and investigated the differences in postprandial glucose kinetics and metabolic response after their consumption. METHODS: Ten healthy male volunteers participated in a randomized, crossover study, consuming 13C-enriched breads with different structures; a control bread (CB) made from wheat flour combined with wheat bran, and a kernel bread (KB) where 85 % of flour was substituted with broken wheat kernels. The structure of the breads was characterized extensively. The use of stable isotopes enabled calculation of glucose kinetics: rate of appearance of exogenous glucose, endogenous glucose production, and glucose clearance rate. Additionally, postprandial plasma concentrations of glucose, insulin, glucagon, incretins, cholecystokinin, and bile acids were analyzed. RESULTS: Despite the attempt to obtain a bread with a low glycemic response by replacing flour by broken kernels, the glycemic response and glucose kinetics were quite similar after consumption of CB and KB. Interestingly, the glucagon-like peptide-1 (GLP-1) response was much lower after KB compared to CB (iAUC, P < 0.005). A clear postprandial increase in plasma conjugated bile acids was observed after both meals. CONCLUSIONS: Substitution of 85 % wheat flour by broken kernels in bread did not result in a difference in glucose response and kinetics, but in a pronounced difference in GLP-1 response. Thus, changing the processing conditions of wheat for baking bread can influence the metabolic response beyond glycemia and may therefore influence health.

Endogenous Glucose Production in Patients With Glycogen Storage Disease Type Ia Estimated by Oral D-[6,6-2H2]-glucose.

CONTEXT: Glycogen storage disease type Ia (GSDIa) is an inborn metabolic disorder characterized by impaired endogenous glucose production (EGP). Monitoring of patients with GSDIa is prioritized because of ongoing treatment developments. Stable isotope tracers may enable reliable EGP monitoring. OBJECTIVE: The aim of this study was to prospectively assess the rate of appearance of endogenous glucose into the bloodstream (Ra) in patients with GSDIa after a single oral D-[6,6-2H2]-glucose dose. METHODS: Ten adult patients with GSDIa and 10 age-, sex-, and body mass index-matched healthy volunteers (HVs) were enrolled. For each participant, 3 oral glucose tracer tests were performed: (1) preprandial/fasted, (2) postprandial, and (3) randomly fed states. Dried blood spots were collected before D-[6,6-2H2]-glucose administration and 10, 20, 30, 40, 50, 60, 75, 90, and 120 minutes thereafter. RESULTS: Glucose Ra in fasted HVs was consistent with previously reported data. The time-averaged glucose Ra was significantly higher in (1) preprandial/fasted patients with GSDIa than HV and (2) postprandial HV compared with fasted HV(P < .05). A progressive decrease in glucose Ra was observed in preprandial/fasted patients with GSDIa; the change in glucose Ra time-course was directly correlated with the change in capillary glucose (P < .05). CONCLUSION: This is the first study to quantify glucose Ra in patients with GSDIa using oral D-[6,6-2H2] glucose. The test can reliably estimate EGP under conditions in which fasting tolerance is unaffected but does not discriminate between relative contributions of EGP (eg, liver, kidney) and exogenous sources (eg, dietary cornstarch). Future application is warranted for longitudinal monitoring after novel genome based treatments in patients with GSDIa in whom nocturnal dietary management can be discontinued.

Changes in bile acid composition are correlated with reduced intestinal cholesterol uptake in intestine-specific WASH-deficient mice.

The Wiskott-Aldrich syndrome protein and SCAR homolog (WASH) complex is a pentameric protein complex localized at endosomes, where it facilitates the transport of numerous receptors from endosomes toward the plasma membrane. Recent studies have shown that the WASH complex plays an essential role in cholesterol and glucose homeostasis in humans and mice. To investigate the physiological importance of intestinal WASH, we ablated the WASH component WASHC1 specifically in murine enterocytes. Male and female intestine-specific WASHC1-deficient mice (Washc1IKO) were challenged with either a standard chow diet or a high-cholesterol (1.25 %) diet (HCD). Washc1IKO mice fed a standard diet did not present any apparent phenotype, but when fed an HCD, their hepatic cholesterol levels were ~ 50 % lower compared to those observed in control mice. The intestinal cholesterol absorption was almost 2-fold decreased in Washc1IKO mice, which translated into increased fecal neutral sterol loss. The intestinal expression of cholesterogenic genes, such as Hmgcs1, Hmgcr, and Ldlr, was significantly higher in Washc1IKO mice than in control mice and correlated with increased whole-body de novo cholesterol synthesis, likely to compensate for impaired intestinal cholesterol absorption. Unexpectedly, the ratio of biliary 12α-/non-12α-hydroxylated bile acids (BAs) was decreased in Washc1IKO mice and reversing this reduced ratio by feeding the mice with the HCD supplemented with 0.5 % (w/w) sodium cholate normalized the improvement of hepatic cholesterol levels in Washc1IKO mice. Our data indicate that the intestinal WASH complex plays an important role in intestinal cholesterol absorption, likely by modulating biliary BA composition.

Intraintestinal fermentation of fructo- and galacto-oligosaccharides and the fate of short-chain fatty acids in humans.

Consumption of fructo- (FOS) and galacto-oligosaccharides (GOS) has health benefits which have been linked in part to short-chain fatty acids (SCFA) production by the gut microbiota. However, detailed knowledge of this process in the human intestine is lacking. We aimed to determine the acute fermentation kinetics of a FOS:GOS mixture in healthy males using a naso-intestinal catheter for sampling directly in the ileum or colon. We studied the fate of SCFA as substrates for glucose and lipid metabolism by the host after infusion of 13C-SCFA. In the human distal ileum, no fermentation of FOS:GOS, nor SCFA production, or bacterial cross-feeding was observed. The relative composition of intestinal microbiota changed rapidly during the test day, which demonstrates the relevance of postprandial intestinal sampling to track acute responses of the microbial community toward interventions. SCFA were vividly taken up and metabolized by the host as shown by incorporation of 13C in various host metabolites.

Pharmacological inhibition of MEK1/2 signaling disrupts bile acid metabolism through loss of Shp and enhanced Cyp7a1 expression.

The RAS-MAPK signaling pathway is one of the most frequently dysregulated pathways in human cancer. Small molecule inhibitors directed against this pathway have clinical activity in patients with various cancer types and can improve patient outcomes. However, the use of these drugs is associated with adverse effects, which can result in dose reduction or treatment interruption. A better molecular understanding of on-target, off-tumor effects may improve toxicity management. In the present study, we aimed to identify early initiating biological changes in the liver upon pharmacological inhibition of the RAS-MAPK signaling pathway. To this end, we tested the effect of MEK inhibitor PD0325901 using mice and human hepatocyte cell lines. Male C57BL/6 mice were treated with either vehicle or PD0325901 for six days, followed by transcriptome analysis of the liver and phenotypic characterization. Pharmacological MEK inhibition altered the expression of 423 genes, of which 78 were upregulated and 345 were downregulated. We identified Shp, a transcriptional repressor, and Cyp7a1, the rate-limiting enzyme in converting cholesterol to bile acids, as the top differentially expressed genes. PD0325901 treatment also affected other genes involved in bile acid regulation, which was associated with changes in the composition of plasma bile acids and composition and total levels of fecal bile acids and elevated predictive biomarkers of early liver toxicity. In conclusion, short-term pharmacological MEK inhibition results in profound changes in bile acid metabolism, which may explain some of the clinical adverse effects of pharmacological inhibition of the RAS-MAPK pathway, including gastrointestinal complications and hepatotoxicity.

Bile Acid Sequestration via Colesevelam Reduces Bile Acid Hydrophobicity and Improves Liver Pathology in Cyp2c70-/- Mice with a Human-like Bile Acid Composition.

Bile acids (BAs) and their signaling pathways have been identified as therapeutic targets for liver and metabolic diseases. We generated Cyp2c70-/- (KO) mice that were not able to convert chenodeoxycholic acid into rodent-specific muricholic acids (MCAs) and, hence, possessed a more hydrophobic, human-like BA pool. Recently, we have shown that KO mice display cholangiopathic features with the development of liver fibrosis. The aim of this study was to determine whether BA sequestration modulates liver pathology in Western type-diet (WTD)-fed KO mice. The BA sequestrant colesevelam was mixed into the WTD (2% w/w) of male Cyp2c70+/+ (WT) and KO mice and the effects were evaluated after 3 weeks of treatment. Colesevelam increased fecal BA excretion in WT and KO mice and reduced the hydrophobicity of biliary BAs in KO mice. Colesevelam ameliorated diet-induced hepatic steatosis in WT mice, whereas KO mice were resistant to diet-induced steatosis and BA sequestration had no additional effects on liver fat content. Total cholesterol concentrations in livers of colesevelam-treated WT and KO mice were significantly lower than those of untreated controls. Of particular note, colesevelam treatment normalized plasma levels of liver damage markers in KO mice and markedly decreased hepatic mRNA levels of fibrogenesis-related genes in KO mice. Lastly, colesevelam did not affect glucose excursions and insulin sensitivity in WT or KO mice. Our data show that BA sequestration ameliorates liver pathology in Cyp2c70-/- mice with a human-like bile acid composition without affecting insulin sensitivity.

Short-term obeticholic acid treatment does not impact cholangiopathy in Cyp2c70-deficient mice with a human-like bile acid composition.

Cyp2c70-/- mice with a human-like bile acid (BA) composition, lacking hydrophilic muricholic acids (MCAs), have been reported to display cholangiopathy and biliary fibrosis with female preponderance that can be reversed by ursodeoxycholic acid (UDCA). Obeticholic acid (OCA), a steroidal BA-like FXR agonist, has been shown to improve liver function in patients with primary biliary cholangitis and is approved as second-line treatment for patients with an inadequate response or intolerance to UDCA. Here, we investigated the impact of OCA on BA hydrophobicity and cholangiopathy in Cyp2c70-/- mice. Male and female wild-type (WT) and Cyp2c70-/- mice were fed a chow diet with or without 10 mg/kg/day OCA for 4 weeks. OCA accounted for 1-5% of biliary BAs, with larger enrichments in Cyp2c70-/- than in WT mice. In WT mice, OCA induced a more hydrophilic, MCA-rich BA pool. In Cyp2c70-/- mice, however, BA pool became more hydrophobic with a larger proportion of chenodeoxycholic acid, attributable to a reduction of BA 12α-hydroxylation. OCA treatment reduced fecal BA excretion, indicating repression of hepatic BA synthesis in both WT and Cyp2c70-/- mice. OCA did, however, not impact on markers of liver (dys)function in plasma nor did it ameliorate cholangiopathy and fibrosis in male or female Cyp2c70-/- mice. OCA treatment also did not affect the expression of genes involved in fibrosis, inflammation and cellular senescence. In conclusion, 4 weeks of OCA treatment oppositely modulates the hydrophobicity of the BA pool in WT and Cyp2c70-/- mice, but does not improve or worsen the characteristic sex-dependent liver pathology in Cyp2c70-/- mice.

A toolbox for the comprehensive analysis of small volume human intestinal samples that can be used with gastrointestinal sampling capsules.

Detailed knowledge on the fate of dietary components inside the human intestinal tract is lacking. Access to this inner world of digestion is now possible through novel human gastrointestinal sampling capsules. Due to the novelty of such devices, no methodology has been published to stabilise and analyse the resulting samples. A complicating factor is that excretion of such capsules in faeces may take days, while degradation of the dietary components continues. Therefore a stabilising reagent should be pre-loaded in the capsule to ensure the measurement of a representative sample. Considering the small volume of recovered samples, analytical methods must be optimized to collect as many data as possible from little material. We present a complete workflow for stabilising and analysing the fermentation status of dietary fibres in such samples, including microbiota, fibre degradation, and short chain fatty acids. The final quenching reagent was designed based on safety and effectiveness to inhibit fructo- and galacto-oligosaccharides degradation and short chain fatty acids production by human ileostomy microbiota, and subsequently validated in faecal samples. The final composition of the stock quenching reagent is 175 mM Tris, 525 mM NaCl, 35 mM EDTA, 12% SDS, and 8 M urea at pH 8.5.

The hepatocyte IKK:NF-κB axis promotes liver steatosis by stimulating de novo lipogenesis and cholesterol synthesis.

OBJECTIVE: Obesity-related chronic inflammation plays an important role in the development of Metabolic Associated Fatty Liver Disease (MAFLD). Although the contribution of the pro-inflammatory NF-κB signaling pathway to the progression from simple steatosis to non-alcoholic steatohepatitis (NASH) is well-established, its role as an initiator of hepatic steatosis and the underlying mechanism remains unclear. Here, we investigated the hypothesis that the hepatocytic NF-κB signaling pathway acts as a metabolic regulator, thereby promoting hepatic steatosis development. METHODS: A murine model expressing a constitutively active form of IKKß in hepatocytes (Hep-IKKßca) was used to activate hepatocyte NF-κB. In addition, IKKßca was also expressed in hepatocyte A20-deficient mice (IKKßca;A20LKO). A20 is an NF-κB-target gene that inhibits the activation of the NF-κB signaling pathway upstream of IKKß. These mouse models were fed a sucrose-rich diet for 8 weeks. Hepatic lipid levels were measured and using [1-13C]-acetate de novo lipogenesis and cholesterol synthesis rate were determined. Gene expression analyses and immunoblotting were used to study the lipogenesis and cholesterol synthesis pathways. RESULTS: Hepatocytic NF-κB activation by expressing IKKßca in hepatocytes resulted in hepatic steatosis without inflammation. Ablation of hepatocyte A20 in Hep-IKKßca mice (IKKßca;A20LKO mice) exacerbated hepatic steatosis, characterized by macrovesicular accumulation of triglycerides and cholesterol, and increased plasma cholesterol levels. Both De novo lipogenesis (DNL) and cholesterol synthesis were found elevated in IKKßca;A20LKO mice. Phosphorylation of AMP-activated kinase (AMPK) - a suppressor in lipogenesis and cholesterol synthesis - was decreased in IKKßca;A20LKO mice. This was paralleled by elevated protein levels of hydroxymethylglutaryl-CoA synthase 1 (HMGCS1) and reduced phosphorylation of HMG-CoA reductase (HMGCR) both key enzymes in the cholesterol synthesis pathway. Whereas inflammation was not observed in young IKKßca;A20LKO mice sustained hepatic NF-κB activation resulted in liver inflammation, together with elevated hepatic and plasma cholesterol levels in middle-aged mice. CONCLUSIONS: The hepatocytic IKK:NF-κB axis is a metabolic regulator by controlling DNL and cholesterol synthesis, independent of its central role in inflammation. The IKK:NF-κB axis controls the phosphorylation levels of AMPK and HMGCR and the protein levels of HMGCS1. Chronic IKK-mediated NF-κB activation may contribute to the initiation of hepatic steatosis and cardiovascular disease risk in MAFLD patients.

Characterization of gut microbial structural variations as determinants of human bile acid metabolism.

Bile acids (BAs) facilitate intestinal fat absorption and act as important signaling molecules in host-gut microbiota crosstalk. BA-metabolizing pathways in the microbial community have been identified, but it remains largely unknown how the highly variable genomes of gut bacteria interact with host BA metabolism. We characterized 8,282 structural variants (SVs) of 55 bacterial species in the gut microbiomes of 1,437 individuals from two cohorts and performed a systematic association study with 39 plasma BA parameters. Both variations in SV-based continuous genetic makeup and discrete clusters showed correlations with BA metabolism. Metagenome-wide association analysis identified 809 replicable associations between bacterial SVs and BAs and SV regulators that mediate the effects of lifestyle factors on BA metabolism. This is the largest microbial genetic association analysis to demonstrate the impact of bacterial SVs on human BA composition, and it highlights the potential of targeting gut microbiota to regulate BA metabolism through lifestyle intervention.