A3907, a systemic ASBT inhibitor, improves cholestasis in mice by multiorgan activity and shows translational relevance to humans.

BACKGROUND AND AIMS: Cholestasis is characterized by intrahepatic accumulation of bile constituents, including bile acids (BAs), which promote liver damage. The apical sodium-dependent BA transporter (ASBT) plays an important role in BA reabsorption and signaling in ileum, bile ducts, and kidneys. Our aim was to investigate the pharmacokinetics and pharmacological activity of A3907, an oral and systemically available ASBT inhibitor in experimental mouse models of cholestasis. In addition, the tolerability, pharmacokinetics, and pharmacodynamics of A3907 were examined in healthy humans. APPROACH AND RESULTS: A3907 was a potent and selective ASBT inhibitor in vitro. In rodents, orally administered A3907 distributed to the ASBT-expressing organs, that is, ileum, liver, and kidneys, and dose dependently increased fecal BA excretion. A3907 improved biochemical, histological, and molecular markers of liver and bile duct injury in Mdr2-/- mice and also had direct protective effects on rat cholangiocytes exposed to cytotoxic BA concentrations in vitro . In bile duct ligated mice, A3907 increased urinary BA elimination, reduced serum BA levels, and prevented body weight loss, while improving markers of liver injury. A3907 was well tolerated and demonstrated target engagement in healthy volunteers. Plasma exposure of A3907 in humans was within the range of systemic concentrations that achieved therapeutic efficacy in mouse. CONCLUSIONS: The systemic ASBT inhibitor A3907 improved experimental cholestatic disease by targeting ASBT function at the intestinal, liver, and kidney levels, resulting in marked clearance of circulating BAs and liver protection. A3907 is well tolerated in humans, supporting further clinical development for the treatment of cholestatic liver diseases.

Of mice and men: murine bile acids explain species differences in the regulation of bile acid and cholesterol metabolism.

Compared with humans, rodents have higher synthesis of cholesterol and bile acids (BAs) and faster clearance and lower levels of serum LDL-cholesterol. Paradoxically, they increase BA synthesis in response to bile duct ligation (BDL). Another difference is the production of hydrophilic 6-hydroxylated muricholic acids (MCAs), which may antagonize the activation of FXRs, in rodents versus humans. We hypothesized that the presence of MCAs is key for many of these metabolic differences between mice and humans. We thus studied the effects of genetic deletion of the Cyp2c70 gene, previously proposed to control MCA formation. Compared with WT animals, KO mice created using the CRISPR/Cas9 system completely lacked MCAs, and displayed >50% reductions in BA and cholesterol synthesis and hepatic LDL receptors, leading to a marked increase in serum LDL-cholesterol. The doubling of BA synthesis following BDL in WT animals was abolished in KO mice, despite extinguished intestinal fibroblast growth factor (Fgf)15 expression in both groups. Accumulation of cholesterol-enriched particles ("Lp-X") in serum was almost eliminated in KO mice. Livers of KO mice were increased 18% in weight, and serum markers of liver function indicated liver damage. The human-like phenotype of BA metabolism in KO mice could not be fully explained by the activation of FXR-mediated changes. In conclusion, the presence of MCAs is critical for many of the known metabolic differences between mice and humans. The Cyp2c70-KO mouse should be useful in studies exploring potential therapeutic targets for human disease.

Gallbladder bile supersaturated with cholesterol in gallstone patients preferentially develops from shortage of bile acids.

Gallstone (GS) formation requires that bile is supersaturated with cholesterol, which is estimated by a cholesterol saturation index (CSI) calculated from gallbladder (GB) total lipids and the mol% (mole percent) of bile acids (BAs), cholesterol, and phospholipids (PLs). Whereas CSI indicates GS risk, we hypothesized that additional comparisons of GB lipid mol% data are inappropriate to identify why CSI is increased in GS disease. We anticipated that GB lipid mmol/l (millimole per liter) levels should instead identify that, and therefore retrieved GB mmol/l data for BAs, cholesterol, and PLs from a study on 145 GS and 87 GS-free patients and compared them with the corresponding mol% data. BA and PL mmol/l levels were 33% and 31% lower in GS patients, while cholesterol was unaltered. CSI was higher in GS patients and correlated inversely with GB levels of BAs and PLs, but not with cholesterol. A literature search confirmed, in 13 studies from 11 countries, that GB BA levels and, to a certain extent, PLs are strongly reduced in GS patients, while cholesterol levels are not elevated. Our findings show that a shortage of BAs is a major reason why GB bile is supersaturated with cholesterol in GS patients. These results are sustainable because they are also valid from a global perspective.

Cholestyramine treatment of healthy humans rapidly induces transient hypertriglyceridemia when treatment is initiated.

Bile acid (BA) production in mice is regulated by hepatic farnesoid X receptors and by intestinal fibroblast growth factor (FGF)-15 (in humans, FGF-19), a suppressor of BA synthesis that also reduces serum triglycerides and glucose. Cholestyramine treatment reduces FGF-19 and induces BA synthesis, whereas plasma triglycerides may increase from unclear reasons. We explored whether FGF-19 may suppress BA synthesis and plasma triglycerides in humans by modulation of FGF-19 levels through long-term cholestyramine treatment at increasing doses. In a second acute experiment, metabolic responses from 1 day of cholestyramine treatment were monitored. Long-term treatment reduced serum FGF-19 by >90%; BA synthesis increased up to 17-fold, whereas serum BAs, triglycerides, glucose, and insulin were stable. After long-term treatment, serum BAs and FGF-19 displayed rebound increases above baseline levels, and BA and cholesterol syntheses normalized after 1 wk without rebound reductions. Acute cholestyramine treatment decreased FGF-19 by 95% overnight and serum BAs by 60%, while BA synthesis increased fourfold and triglycerides doubled. The results support that FGF-19 represses BA synthesis but not serum triglycerides. However, after cessation of both long-term and 1-day cholestyramine treatment, circulating FGF-19 levels were normalized within 2 days, whereas BA synthesis remained significantly induced in both situations, indicating that also other mechanisms than the FGF-19 pathway are responsible for stimulation of BA synthesis elicited by cholestyramine. Several of the responses during cholestyramine treatment persisted at least 6 days after treatment, highlighting the importance of removing such treatment well before evaluating dynamics of the enterohepatic circulation in humans.

A Physiology-Based Model of Bile Acid Distribution and Metabolism Under Healthy and Pathologic Conditions in Human Beings.

BACKGROUND & AIMS: Disturbances of the enterohepatic circulation of bile acids (BAs) are seen in a number of clinically important conditions, including metabolic disorders, hepatic impairment, diarrhea, and gallstone disease. To facilitate the exploration of underlying pathogenic mechanisms, we developed a mathematical model built on quantitative physiological observations across different organs. METHODS: The model consists of a set of kinetic equations describing the syntheses of cholic, chenodeoxycholic, and deoxycholic acids, as well as time-related changes of their respective free and conjugated forms in the systemic circulation, the hepatoportal region, and the gastrointestinal tract. The core structure of the model was adapted from previous modeling research and updated based on recent mechanistic insights, including farnesoid X receptor-mediated autoregulation of BA synthesis and selective transport mechanisms. The model was calibrated against existing data on BA distribution and feedback regulation. RESULTS: According to model-based predictions, changes in intestinal motility, BA absorption, and biotransformation rates affected BA composition and distribution differently, as follows: (1) inhibition of transintestinal BA flux (eg, in patients with BA malabsorption) or acceleration of intestinal motility, followed by farnesoid X receptor down-regulation, was associated with colonic BA accumulation; (2) in contrast, modulation of the colonic absorption process was predicted to not affect the BA pool significantly; and (3) activation of ileal deconjugation (eg, in patents with small intestinal bacterial overgrowth) was associated with an increase in the BA pool, owing to higher ileal permeability of unconjugated BA species. CONCLUSIONS: This model will be useful in further studying how BA enterohepatic circulation modulation may be exploited for therapeutic benefits.

Energy restriction in obese women suggest linear reduction of hepatic fat content and time-dependent metabolic improvements.

Energy restriction reduces liver fat, improves hepatic insulin resistance and lipid metabolism. However, temporal data in which these metabolic improvements occur and their interplay is incomplete. By performing repeated MRI scans and blood analysis at day 0, 3, 7, 14 and 28 the temporal changes in liver fat and related metabolic factors were assessed at five times during a low-calorie diet (LCD, 800-1100 kcal/day) in ten obese non-diabetic women (BMI 41.7 ± 2.6 kg/m2) whereof 6 had NAFLD. Mean weight loss was 7.4 ± 1.2 kg (0.7 kg/day) and liver fat decreased by 51 ± 16%, resulting in only three subjects having NAFLD at day 28. Marked alteration of insulin, NEFA, ALT and 3-hydroxybuturate was evident 3 days after commencing LCD, whereas liver fat showed a moderate but a linear reduction across the 28 days. Other circulating-liver fat markers (e.g. triglycerides, adiponectin, stearoyl-CoA desaturase-1 index, fibroblast growth factor 21) demonstrated modest and variable changes. Marked elevations of NEFA, 3-hydroxybuturate and ALT concentrations occurred until day 14, likely reflecting increased tissue lipolysis, fat oxidation and upregulated hepatic fatty acid oxidation. In summary, these results suggest linear reduction in liver fat, time-specific changes in metabolic markers and insulin resistance in response to energy restriction.

Overeating Saturated Fat Promotes Fatty Liver and Ceramides Compared With Polyunsaturated Fat: A Randomized Trial.

CONTEXT: Saturated fatty acid (SFA) vs polyunsaturated fatty acid (PUFA) may promote nonalcoholic fatty liver disease by yet unclear mechanisms. OBJECTIVE: To investigate if overeating SFA- and PUFA-enriched diets lead to differential liver fat accumulation in overweight and obese humans. DESIGN: Double-blind randomized trial (LIPOGAIN-2). Overfeeding SFA vs PUFA for 8 weeks, followed by 4 weeks of caloric restriction. SETTING: General community. PARTICIPANTS: Men and women who are overweight or have obesity (n = 61). INTERVENTION: Muffins, high in either palm (SFA) or sunflower oil (PUFA), were added to the habitual diet. MAIN OUTCOME MEASURES: Lean tissue mass (not reported here). Secondary and exploratory outcomes included liver and ectopic fat depots. RESULTS: By design, body weight gain was similar in SFA (2.31 ± 1.38 kg) and PUFA (2.01 ± 1.90 kg) groups, P = 0.50. SFA markedly induced liver fat content (50% relative increase) along with liver enzymes and atherogenic serum lipids. In contrast, despite similar weight gain, PUFA did not increase liver fat or liver enzymes or cause any adverse effects on blood lipids. SFA had no differential effect on the accumulation of visceral fat, pancreas fat, or total body fat compared with PUFA. SFA consistently increased, whereas PUFA reduced circulating ceramides, changes that were moderately associated with liver fat changes and proposed markers of hepatic lipogenesis. The adverse metabolic effects of SFA were reversed by calorie restriction. CONCLUSIONS: SFA markedly induces liver fat and serum ceramides, whereas dietary PUFA prevents liver fat accumulation and reduces ceramides and hyperlipidemia during excess energy intake and weight gain in overweight individuals.

Liver macrophages regulate systemic metabolism through non-inflammatory factors.

Liver macrophages (LMs) have been proposed to contribute to metabolic disease through secretion of inflammatory cytokines. However, anti-inflammatory drugs lead to only modest improvements in systemic metabolism. Here we show that LMs do not undergo a proinflammatory phenotypic switch in obesity-induced insulin resistance in flies, mice and humans. Instead, we find that LMs produce non-inflammatory factors, such as insulin-like growth factor-binding protein 7 (IGFBP7), that directly regulate liver metabolism. IGFBP7 binds to the insulin receptor and induces lipogenesis and gluconeogenesis via activation of extracellular-signal-regulated kinase (ERK) signalling. We further show that IGFBP7 is subject to RNA editing at a higher frequency in insulin-resistant than in insulin-sensitive obese patients (90% versus 30%, respectively), resulting in an IGFBP7 isoform with potentially higher capacity to bind to the insulin receptor. Our study demonstrates that LMs can contribute to insulin resistance independently of their inflammatory status and indicates that non-inflammatory factors produced by macrophages might represent new drug targets for the treatment of metabolic diseases.

Author Correction: Liver macrophages regulate systemic metabolism through non-inflammatory factors.

In the version of this article initially published, author Volker M. Lauschke had affiliation number 13; the correct affiliation number is 12. The error has been corrected in the HTML and PDF versions of the article.

Deficiency and supplementation of PUFA in the diet have similar effects on the age-associated changes in rat-plasma cholesterol levels.

Levels of plasma cholesterol, particularly LDL cholesterol, increase with increasing age in humans and rodents. Feeding a fish oil-rich diet may exert hypocholesterolemic effects. The aim of this work was to examine the effects of a life-long administration of a PUFA-enriched diet and of a PUFA-deficient diet in male Sprague-Dawley rats on the age-associated increases in plasma cholesterol and triglycerides. Diet had small effects on body-weight, and had dramatic effects on liver phospholipids-fatty acids. Surprisingly, both diets counteracted the age-associated changes in plasma cholesterol and triglycerides similarly and benefits were already visible in adult rats.

Omega-3 as well as caloric restriction prevent the age-related modifications of cholesterol metabolism.

Intracellular concentration of cholesterol is regulated by the balance between endogenous synthesis and exogenous uptake; endogenous synthesis is subject to feedback control of hepatic 3-hydroxy-3-methyl-glutaryl-CoA reductase activity, while the exogenous supply is mainly controlled by the modulation of the low-density lipoprotein receptor. During ageing, hepatic lipid modifications occur and caloric restriction are able to prevent these changes. So, the aim of this work was to evaluate the mechanisms underlying the effect exerted both by caloric restrictions and by a diet enriched with Omega-3 fatty acids, on the cholesterol plasma levels during ageing, by studying the regulation of the protein involved in cholesterol homeostasis maintenance. Livers from diet restricted and Omega-3 supplemented diet fed 24-month-old rat were used to analyze, the protein complex of cholesterol homeostasis maintenance and those ones that are able to modulate 3-hydroxy-3-methyl-glutaryl-CoA reductase. The data obtained demonstrate that both caloric restriction and Omega-3 supplemented diets are able to prevent hypercholesterolemia, by regulating HMG-CoAR activation state by controlling ROS production and p38 phosphorylation. Moreover also the age-dependent loss of LDLr membrane exposition is prevented.

Potential role of milk fat globule membrane in modulating plasma lipoproteins, gene expression, and cholesterol metabolism in humans: a randomized study.

BACKGROUND: Butter is rich in saturated fat [saturated fatty acids (SFAs)] and can increase plasma low density lipoprotein (LDL) cholesterol, which is a major risk factor for cardiovascular disease. However, compared with other dairy foods, butter is low in milk fat globule membrane (MFGM) content, which encloses the fat. We hypothesized that different dairy foods may have distinct effects on plasma lipids because of a varying content of MFGM. OBJECTIVE: We aimed to investigate whether the effects of milk fat on plasma lipids and cardiometabolic risk markers are modulated by the MFGM content. DESIGN: The study was an 8-wk, single-blind, randomized, controlled isocaloric trial with 2 parallel groups including overweight men and women (n = 57 randomly assigned). For the intervention, subjects consumed 40 g milk fat/d as either whipping cream (MFGM diet) or butter oil (control diet). Intervention foods were matched for total fat, protein, carbohydrates, and calcium. Subjects were discouraged from consuming any other dairy products during the study. Plasma markers of cholesterol absorption and hepatic cholesterol metabolism were assessed together with global gene-expression analyses in peripheral blood mononuclear cells. RESULTS: As expected, the control diet increased plasma lipids, whereas the MFGM diet did not [total cholesterol (±SD): +0.30 ± 0.49 compared with -0.04 ± 0.49 mmol/L, respectively (P = 0.024); LDL cholesterol: +0.36 ± 0.50 compared with +0.04 ± 0.36 mmol/L, respectively (P = 0.024); apolipoprotein B:apolipoprotein A-I ratio: +0.03 ± 0.09 compared with -0.05 ± 0.10 mmol/L, respectively (P = 0.007); and non-HDL cholesterol: +0.24 ± 0.49 compared with -0.14 ± 0.51 mmol/L, respectively (P = 0.013)]. HDL-cholesterol, triglyceride, sitosterol, lathosterol, campesterol, and proprotein convertase subtilisin/kexin type 9 plasma concentrations and fatty acid compositions did not differ between groups. Nineteen genes were differentially regulated between groups, and these genes were mostly correlated with lipid changes. CONCLUSIONS: In contrast to milk fat without MFGM, milk fat enclosed by MFGM does not impair the lipoprotein profile. The mechanism is not clear although suppressed gene expression by MFGM correlated inversely with plasma lipids. The food matrix should be considered when evaluating cardiovascular aspects of different dairy foods. This trial was registered at clinicaltrials.gov as NCT01767077.

Stimulation of autophagy by antilipolytic drugs may rescue rodents from age-associated hypercholesterolemia.

Aging is characterized by several metabolic changes responsible for the decline of certain functions and the appearance of age-related diseases, including hypercholesterolemia, which is the main risk factor for atherosclerosis and cardiovascular disease. Similar changes in a number of morphological and biochemical parameters were observed in rats. Caloric restriction (CR) was shown to increase longevity and prevent age-related diseases in various organisms, and to counteract the age-associated increase in plasma cholesterol. CR was thought to operate through the stimulation of the process of macroautophagy. The aim of this work was to investigate the effect of the stimulation of macroautophagy on age-associated cholesterolemia. Mature Sprague-Dawley rats were fasted overnight and given the antilipolytic agent 3,5-dimethylpyrazole (DMP; 12 mg/kg b.w. in 0.2 mL of saline, intraperitoneally). The age-related changes in cholesterol plasma level, 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMG-CoA-R) activity, and lipoperoxidation were determined. Low-density lipoprotein (LDL) receptor expression was determined by immunoblot of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)-separated liver membranes. Results show that the stimulation of macroautophagy reduces the total LDL and high-density lipoprotein (HDL) cholesterol plasma level to juvenile values, and triglycerides levels even lower. The hypocholesterolemic action of DMP requires neither the counteraction of the age-related changes in the HMG-CoA-R activation state and regulation, nor the counteraction of the age-related increase in lipoperoxidation, and only involves a restoration of the numbers of LDL receptors on liver membranes to juvenile levels.