Feeding diversified protein sources exacerbates hepatic insulin resistance via increased gut microbial branched-chain fatty acids and mTORC1 signaling in obese mice.

Animal models of human diseases are classically fed purified diets that contain casein as the unique protein source. We show that provision of a mixed protein source mirroring that found in the western diet exacerbates diet-induced obesity and insulin resistance by potentiating hepatic mTORC1/S6K1 signaling as compared to casein alone. These effects involve alterations in gut microbiota as shown by fecal microbiota transplantation studies. The detrimental impact of the mixed protein source is also linked with early changes in microbial production of branched-chain fatty acids (BCFA) and elevated plasma and hepatic acylcarnitines, indicative of aberrant mitochondrial fatty acid oxidation. We further show that the BCFA, isobutyric and isovaleric acid, increase glucose production and activate mTORC1/S6K1 in hepatocytes. Our findings demonstrate that alteration of dietary protein source exerts a rapid and robust impact on gut microbiota and BCFA with significant consequences for the development of obesity and insulin resistance.

Treatment with HIV-Protease Inhibitor Nelfinavir Identifies Membrane Lipid Composition and Fluidity as a Therapeutic Target in Advanced Multiple Myeloma.

The HIV-protease inhibitor nelfinavir has shown broad anticancer activity in various preclinical and clinical contexts. In patients with advanced, proteasome inhibitor (PI)-refractory multiple myeloma, nelfinavir-based therapy resulted in 65% partial response or better, suggesting that this may be a highly active chemotherapeutic option in this setting. The broad anticancer mechanism of action of nelfinavir implies that it interferes with fundamental aspects of cancer cell biology. We combined proteome-wide affinity-purification of nelfinavir-interacting proteins with genome-wide CRISPR/Cas9-based screening to identify protein partners that interact with nelfinavir in an activity-dependent manner alongside candidate genetic contributors affecting nelfinavir cytotoxicity. Nelfinavir had multiple activity-specific binding partners embedded in lipid bilayers of mitochondria and the endoplasmic reticulum. Nelfinavir affected the fluidity and composition of lipid-rich membranes, disrupted mitochondrial respiration, blocked vesicular transport, and affected the function of membrane-embedded drug efflux transporter ABCB1, triggering the integrated stress response. Sensitivity to nelfinavir was dependent on ADIPOR2, which maintains membrane fluidity by promoting fatty acid desaturation and incorporation into phospholipids. Supplementation with fatty acids prevented the nelfinavir-induced effect on mitochondrial metabolism, drug-efflux transporters, and stress-response activation. Conversely, depletion of fatty acids/cholesterol pools by the FDA-approved drug ezetimibe showed a synergistic anticancer activity with nelfinavir in vitro. These results identify the modification of lipid-rich membranes by nelfinavir as a novel mechanism of action to achieve broad anticancer activity, which may be suitable for the treatment of PI-refractory multiple myeloma. SIGNIFICANCE: Nelfinavir induces lipid bilayer stress in cellular organelles that disrupts mitochondrial respiration and transmembrane protein transport, resulting in broad anticancer activity via metabolic rewiring and activation of the unfolded protein response.

Association of dietary and gut microbiota-related metabolites with calcific aortic stenosis.

BACKGROUND: Histopathological changes in calcific aortic stenosis (CAS) resemble changes in coronary atherosclerosis. Concerning recent evidence on dietary and gut microbiota-related metabolites representing players in atherosclerosis, we aimed to investigate the link between dietary and gut microbiota-derived metabolites and CAS. METHODS: We consecutively recruited eligible subjects with moderate-severe CAS (n = 60), aortic sclerosis (ASc) (n = 49) and age and gender-matched control subjects (n = 48) in May 2016-December 2016. Plasma dietary and gut microbiota-related metabolite levels, namely choline, betaine, and trimethylamine N-oxide (TMAO), were measured using ultra-performance liquid chromatography-tandem mass spectroscopy method. Histopathological examinations were performed in patients that underwent aortic valve surgery. RESULTS: Prevalence of traditional cardiovascular risk factors or co-morbidities did not differ among groups (all p > 0.05). CAS patients had higher plasma choline levels compared to both control (p < 0.001) and ASc (p = 0.006). Plasma betaine and TMAO levels were similar (both p > 0.05). Compared to the lowest quartile choline levels (<11.15 µM), patients with the highest quartile choline levels (≥14.98 µM) had higher aortic valvular (p < 0.001) and mitral annular (p = 0.013) calcification scores. Plasma choline levels were independently associated with aortic peak flow velocity (B ± SE:0.165 ± 0.060, p = 0.009). Choline levels were elevated in subjects who had aortic valves with denser lymphocyte infiltration (p < 0.001), neovascularization (p = 0.011), osseous metaplasia (p = 0.004), more severe tissue remodelling (p = 0.002) and calcification (p = 0.002). CONCLUSION: We found a significant association between choline levels and CAS presence and severity depicted on imaging modalities and histopathological examinations. Our study may open new horizons for prevention of CAS.

Lipid profiling of human diabetic myocardium reveals differences in triglyceride fatty acyl chain length and degree of saturation.

BACKGROUND: Type 2 diabetes is a major health problem in the world, and is strongly associated with impaired cardiac function and increased mortality. The causal relationship between type 2 diabetes and impaired cardiac function is still incompletely understood but changes in the cardiac lipid metabolism are believed to be a contributing factor. The objective of this study was to determine the lipid profile in human myocardial biopsies collected in vivo from patients with type 2 diabetes and compare to non-diabetic controls. METHOD: We conducted full lipidomics analyses, using mass spectrometry, of 85 right atrial biopsies obtained from diabetic and non-diabetic patients undergoing elective cardiac surgery. The patients were characterized clinically and serum was analyzed for lipids and biochemical markers. RESULTS: The groups did not differ in BMI and in circulating triglycerides. We demonstrate that type 2 diabetes is associated with alterations in the cardiac lipidome. Interestingly, the absolute amount of lipids is not altered in the diabetic myocardium. However, triglycerides with longer fatty acyl chains are more abundant and there is a higher degree of unsaturated fatty acid chains in triglycerides in diabetic myocardium. CONCLUSIONS: Our study reveals that type 2 diabetes is a relatively strong determinant of the human cardiac lipidome (compared to other clinical variables). Although the total lipid content in the diabetic myocardium is not increased, the lipid composition is markedly affected.

The acute effect of metabolic cofactor supplementation: a potential therapeutic strategy against non-alcoholic fatty liver disease.

The prevalence of non-alcoholic fatty liver disease (NAFLD) continues to increase dramatically, and there is no approved medication for its treatment. Recently, we predicted the underlying molecular mechanisms involved in the progression of NAFLD using network analysis and identified metabolic cofactors that might be beneficial as supplements to decrease human liver fat. Here, we first assessed the tolerability of the combined metabolic cofactors including l-serine, N-acetyl-l-cysteine (NAC), nicotinamide riboside (NR), and l-carnitine by performing a 7-day rat toxicology study. Second, we performed a human calibration study by supplementing combined metabolic cofactors and a control study to study the kinetics of these metabolites in the plasma of healthy subjects with and without supplementation. We measured clinical parameters and observed no immediate side effects. Next, we generated plasma metabolomics and inflammatory protein markers data to reveal the acute changes associated with the supplementation of the metabolic cofactors. We also integrated metabolomics data using personalized genome-scale metabolic modeling and observed that such supplementation significantly affects the global human lipid, amino acid, and antioxidant metabolism. Finally, we predicted blood concentrations of these compounds during daily long-term supplementation by generating an ordinary differential equation model and liver concentrations of serine by generating a pharmacokinetic model and finally adjusted the doses of individual metabolic cofactors for future human clinical trials.

Impairment of bile acid metabolism by perfluorooctanoic acid (PFOA) and perfluorooctanesulfonic acid (PFOS) in human HepaRG hepatoma cells.

Perfluorooctanoic acid (PFOA) and perfluorooctanesulfonic acid (PFOS) are man-made chemicals that are used for the fabrication of many products with water- and dirt-repellent properties. The toxicological potential of both substances is currently under debate. In a recent Scientific Opinion, the European Food Safety Authority (EFSA) has identified increased serum total cholesterol levels in humans as one major critical effect being associated with exposure to PFOA or PFOS. In animal studies, both substances induced a decrease of serum cholesterol levels, and the underlying molecular mechanism(s) for these opposed effects are unclear so far. In the present study, we examined the impact of PFOA and PFOS on cholesterol homoeostasis in the human HepaRG cell line as a model for human hepatocytes. Cholesterol levels in HepaRG cells were not affected by PFOA or PFOS, but both substances strongly decreased synthesis of a number of bile acids. The expression of numerous genes whose products are involved in synthesis, metabolism and transport of cholesterol and bile acids was strongly affected by PFOA and PFOS at concentrations above 10 µM. Notably, both substances led to a strong decrease of CYP7A1, the key enzyme catalyzing the rate-limiting step in the synthesis of bile acids from cholesterol, both at the protein level and at the level of gene expression. Moreover, both substances led to a dilatation of bile canaliculi that are formed by differentiated HepaRG cells in vitro. Similar morphological changes are known to be induced by cholestatic agents in vivo. Thus, the strong impact of PFOA and PFOS on bile acid synthesis and bile canalicular morphology in our in vitro experiments may allow the notion that both substances have a cholestatic potential that is connected to the observed increased serum cholesterol levels in humans in epidemiological studies.

Donor metabolic characteristics drive effects of faecal microbiota transplantation on recipient insulin sensitivity, energy expenditure and intestinal transit time.

OBJECTIVE: Bariatric surgery improves glucose metabolism. Recent data suggest that faecal microbiota transplantation (FMT) using faeces from postbariatric surgery diet-induced obese mice in germ-free mice improves glucose metabolism and intestinal homeostasis. We here investigated whether allogenic FMT using faeces from post-Roux-en-Y gastric bypass donors (RYGB-D) compared with using faeces from metabolic syndrome donors (METS-D) has short-term effects on glucose metabolism, intestinal transit time and adipose tissue inflammation in treatment-naïve, obese, insulin-resistant male subjects. DESIGN: Subjects with metabolic syndrome (n=22) received allogenic FMT either from RYGB-D or METS-D. Hepatic and peripheral insulin sensitivity as well as lipolysis were measured at baseline and 2 weeks after FMT by hyperinsulinaemic euglycaemic stable isotope (2H2-glucose and 2H5-glycerol) clamp. Secondary outcome parameters were changes in resting energy expenditure, intestinal transit time, faecal short-chain fatty acids (SCFA) and bile acids, and inflammatory markers in subcutaneous adipose tissue related to intestinal microbiota composition. Faecal SCFA, bile acids, glycaemic control and inflammatory parameters were also evaluated at 8 weeks. RESULTS: We observed a significant decrease in insulin sensitivity 2 weeks after allogenic METS-D FMT (median rate of glucose disappearance: from 40.6 to 34.0 µmol/kg/min; p<0.01). Moreover, a trend (p=0.052) towards faster intestinal transit time following RYGB-D FMT was seen. Finally, we observed changes in faecal bile acids (increased lithocholic, deoxycholic and (iso)lithocholic acid after METS-D FMT), inflammatory markers (decreased adipose tissue chemokine ligand 2 (CCL2) gene expression and plasma CCL2 after RYGB-D FMT) and changes in several intestinal microbiota taxa. CONCLUSION: Allogenic FMT using METS-D decreases insulin sensitivity in metabolic syndrome recipients when compared with using post-RYGB-D. Further research is needed to delineate the role of donor characteristics in FMT efficacy in human insulin-resistant subjects. TRIAL REGISTRATION NUMBER: NTR4327.

Associations between Dietary Patterns and Bile Acids-Results from a Cross-Sectional Study in Vegans and Omnivores.

Bile acids play an active role in fat metabolism and, in high-fat diets, elevated concentrations of fecal bile acids may be related to an increased risk of colorectal cancer. This study investigated concentrations of fecal and serum bile acids in 36 vegans and 36 omnivores. The reduced rank regression was used to identify dietary patterns associated with fecal bile acids. Dietary patterns were derived with secondary and conjugated fecal bile acids as response variables and 53 food groups as predictors. Vegans had higher fiber (p < 0.01) and lower fat (p = 0.0024) intake than omnivores. In serum, primary and glycine-conjugated bile acids were higher in vegans than in omnivores (p ≤ 0.01). All fecal bile acids were significantly lower in vegans compared to omnivores (p < 0.01). Processed meat, fried potatoes, fish, margarine, and coffee contributed most positively, whereas muesli most negatively to a dietary pattern that was directly associated with all fecal bile acids. According to the pattern, fat intake was positively and fiber intake was inversely correlated with bile acids. The findings contribute to the evidence that, in particular, animal products and fat may play a part in higher levels of fecal bile acids.

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.

Obeticholic acid may increase the risk of gallstone formation in susceptible patients.

BACKGROUND & AIMS: The nuclear farnesoid X receptor (FXR) agonist obeticholic acid (OCA) has been developed for the treatment of liver diseases. We aimed to determine whether OCA treatment increases the risk of gallstone formation. METHODS: Twenty patients awaiting laparoscopic cholecystectomy were randomized to treatment with OCA (25 mg/day) or placebo for 3 weeks until the day before surgery. Serum bile acids (BAs), the BA synthesis marker C4 (7α-hydroxy-4-cholesten-3-one), and fibroblast growth factor 19 (FGF19) were measured before and after treatment. During surgery, biopsies from the liver and the whole bile-filled gallbladder were collected for analyses of gene expression, biliary lipids and FGF19. RESULTS: In serum, OCA increased FGF19 (from 95.0 ±â€¯8.5 to 234.4 ±â€¯35.6 ng/L) and decreased C4 (from 31.4 ±â€¯22.8 to 2.8 ±â€¯4.0 nmol/L) and endogenous BAs (from 1,312.2 ±â€¯236.2 to 517.7 ±â€¯178.9 nmol/L; all p <0.05). At surgery, BAs in gallbladder bile were lower in patients that received OCA than in controls (OCA, 77.9 ±â€¯53.6 mmol/L; placebo, 196.4 ±â€¯99.3 mmol/L; p <0.01), resulting in a higher cholesterol saturation index (OCA, 2.8 ±â€¯1.1; placebo, 1.8 ±â€¯0.8; p <0.05). In addition, hydrophobic OCA conjugates accounted for 13.6 ±â€¯5.0% of gallbladder BAs after OCA treatment, resulting in a higher hydrophobicity index (OCA, 0.43 ±â€¯0.09; placebo, 0.34 ±â€¯0.07, p <0.05). Gallbladder FGF19 levels were 3-fold higher in OCA patients than in controls (OCA, 40.3 ±â€¯16.5 ng/L; placebo, 13.5 ±â€¯13.1 ng/ml; p <0.005). Gene expression analysis indicated that FGF19 mainly originated from the gallbladder epithelium. CONCLUSIONS: Our results show for the first time an enrichment of FGF19 in human bile after OCA treatment. In accordance with its murine homolog FGF15, FGF19 might trigger relaxation and filling of the gallbladder which, in combination with increased cholesterol saturation and BA hydrophobicity, would enhance the risk of gallstone development. LAY SUMMARY: Obeticholic acid increased human gallbladder cholesterol saturation and bile acid hydrophobicity, both decreasing cholesterol solubility in bile. Together with increased hepatobiliary levels of fibroblast growth factor 19, our findings suggest that pharmacological activation of the farnesoid X receptor increases the risk of gallstone formation. Clinical trial number: NCT01625026.

AdipoR1 and AdipoR2 maintain membrane fluidity in most human cell types and independently of adiponectin.

The FA composition of phospholipids must be tightly regulated to maintain optimal cell membrane properties and compensate for a highly variable supply of dietary FAs. Previous studies have shown that AdipoR2 and its homologue PAQR-2 are important regulators of phospholipid FA composition in HEK293 cells and Caenorhabditiselegans, respectively. Here we show that both AdipoR1 and AdipoR2 are essential for sustaining desaturase expression and high levels of unsaturated FAs in membrane phospholipids of many human cell types, including primary human umbilical vein endothelial cells, and for preventing membrane rigidification in cells challenged with exogenous palmitate, a saturated FA. Three independent methods confirm the role of the AdipoRs as regulators of membrane composition and fluidity: fluorescence recovery after photobleaching, measurements of Laurdan dye generalized polarization, and mass spectrometry to determine the FA composition of phospholipids. Furthermore, we show that the AdipoRs can prevent lipotoxicity in the complete absence of adiponectin, their putative ligand. We propose that the primary cellular function of AdipoR1 and AdipoR2 is to maintain membrane fluidity in most human cell types and that adiponectin is not required for this function.

Pyruvate kinase L/R is a regulator of lipid metabolism and mitochondrial function.

The pathogenesis of non-alcoholic fatty liver disease (NAFLD) and hepatocellular carcinoma (HCC) has been associated with altered expression of liver-specific genes including pyruvate kinase liver and red blood cell (PKLR), patatin-like phospholipase domain containing 3 (PNPLA3) and proprotein convertase subtilisin/kexin type 9 (PCSK9). Here, we inhibited and overexpressed the expression of these three genes in HepG2 cells, generated RNA-seq data before and after perturbation and revealed the altered global biological functions with the modulation of these genes using integrated network (IN) analysis. We found that modulation of these genes effects the total triglycerides levels within the cells and viability of the cells. Next, we generated IN for HepG2 cells, identified reporter transcription factors based on IN and found that the modulation of these genes affects key metabolic pathways associated with lipid metabolism (steroid biosynthesis, PPAR signalling pathway, fatty acid synthesis and oxidation) and cancer development (DNA replication, cell cycle and p53 signalling) involved in the progression of NAFLD and HCC. Finally, we observed that inhibition of PKLR lead to decreased glucose uptake and decreased mitochondrial activity in HepG2 cells. Hence, our systems level analysis indicated that PKLR can be targeted for development efficient treatment strategy for NAFLD and HCC.

Vimentin deficiency in macrophages induces increased oxidative stress and vascular inflammation but attenuates atherosclerosis in mice.

The aim was to clarify the role of vimentin, an intermediate filament protein abundantly expressed in activated macrophages and foam cells, in macrophages during atherogenesis. Global gene expression, lipid uptake, ROS, and inflammation were analyzed in bone-marrow derived macrophages from vimentin-deficient (Vim-/-) and wild-type (Vim+/+) mice. Atherosclerosis was induced in Ldlr-/- mice transplanted with Vim-/- and Vim+/+ bone marrow, and in Vim-/- and Vim+/+ mice injected with a PCSK9 gain-of-function virus. The mice were fed an atherogenic diet for 12-15 weeks. We observed impaired uptake of native LDL but increased uptake of oxLDL in Vim-/- macrophages. FACS analysis revealed increased surface expression of the scavenger receptor CD36 on Vim-/- macrophages. Vim-/- macrophages also displayed increased markers of oxidative stress, activity of the transcription factor NF-κB, secretion of proinflammatory cytokines and GLUT1-mediated glucose uptake. Vim-/- mice displayed decreased atherogenesis despite increased vascular inflammation and increased CD36 expression on macrophages in two mouse models of atherosclerosis. We demonstrate that vimentin has a strong suppressive effect on oxidative stress and that Vim-/- mice display increased vascular inflammation with increased CD36 expression on macrophages despite decreased subendothelial lipid accumulation. Thus, vimentin has a key role in regulating inflammation in macrophages during atherogenesis.

Lanosterol Synthase Regulates Human Rhinovirus Replication in Human Bronchial Epithelial Cells.

Human rhinovirus (RV) infections are a significant risk factor for exacerbations of asthma and chronic obstructive pulmonary disease. Thus, approaches to prevent RV infection in such patients would give significant benefit. Through RNA interference library screening, we identified lanosterol synthase (LSS), a component of the cholesterol biosynthetic pathway, as a novel regulator of RV replication in primary normal human bronchial epithelial cells. Selective knock down of LSS mRNA with short interfering RNA inhibited RV2 replication in normal human bronchial epithelial cells. Small molecule inhibitors of LSS mimicked the effect of LSS mRNA knockdown in a concentration-dependent manner. We further demonstrated that the antiviral effect is not dependent on a reduction in total cellular cholesterol but requires a 24-hour preincubation with the LSS inhibitor. The rank order of antiviral potency of the LSS inhibitors used was consistent with LSS inhibition potency; however, all compounds showed remarkably higher potency against RV compared with the LSS enzyme potency. We showed that LSS inhibition led to an induction of 24(S),25 epoxycholesterol, an important regulator of the sterol pathway. We also demonstrated that LSS inhibition led to a profound increase in expression of the innate antiviral defense protein, IFN-ß. We found LSS to be a novel regulator of RV replication and innate antiviral immunity and identified a potential molecular mechanism for this effect, via induction of 24(S),25 epoxycholesterol. Inhibition of LSS could therefore be a novel therapeutic target for prevention of RV-induced exacerbations.

An Integrated Understanding of the Rapid Metabolic Benefits of a Carbohydrate-Restricted Diet on Hepatic Steatosis in Humans.

A carbohydrate-restricted diet is a widely recommended intervention for non-alcoholic fatty liver disease (NAFLD), but a systematic perspective on the multiple benefits of this diet is lacking. Here, we performed a short-term intervention with an isocaloric low-carbohydrate diet with increased protein content in obese subjects with NAFLD and characterized the resulting alterations in metabolism and the gut microbiota using a multi-omics approach. We observed rapid and dramatic reductions of liver fat and other cardiometabolic risk factors paralleled by (1) marked decreases in hepatic de novo lipogenesis; (2) large increases in serum ß-hydroxybutyrate concentrations, reflecting increased mitochondrial ß-oxidation; and (3) rapid increases in folate-producing Streptococcus and serum folate concentrations. Liver transcriptomic analysis on biopsy samples from a second cohort revealed downregulation of the fatty acid synthesis pathway and upregulation of folate-mediated one-carbon metabolism and fatty acid oxidation pathways. Our results highlight the potential of exploring diet-microbiota interactions for treating NAFLD.

Serine/threonine protein kinase 25 antisense oligonucleotide treatment reverses glucose intolerance, insulin resistance, and nonalcoholic fatty liver disease in mice.

Nonalcoholic fatty liver disease (NAFLD) contributes to the pathogenesis of type 2 diabetes and cardiovascular disease, and patients with nonalcoholic steatohepatitis (NASH) are also at risk of developing cirrhosis, liver failure, and hepatocellular carcinoma. To date, no specific therapy exists for NAFLD/NASH, which has been recognized as one of the major unmet medical needs of the twenty-first century. We recently identified serine/threonine protein kinase (STK)25 as a critical regulator of energy homeostasis and NAFLD progression. Here, we investigated the effect of antisense oligonucleotides (ASOs) targeting Stk25 on the metabolic and molecular phenotype of mice after chronic exposure to dietary lipids. We found that Stk25 ASOs efficiently reversed high-fat diet-induced systemic hyperglycemia and hyperinsulinemia, improved whole-body glucose tolerance and insulin sensitivity, and ameliorated liver steatosis, inflammatory infiltration, apoptosis, hepatic stellate cell activation, and nutritional fibrosis in obese mice. Moreover, Stk25 ASOs suppressed the abundance of liver acetyl-coenzyme A carboxylase (ACC) protein, a key regulator of both lipid oxidation and synthesis, revealing the likely mechanism underlying repression of hepatic fat accumulation by ASO treatment. We also found that STK25 protein levels correlate significantly and positively with NASH development in human liver biopsies, and several common nonlinked single-nucleotide polymorphisms in the human STK25 gene are associated with altered liver fat, supporting a critical role of STK25 in the pathogenesis of NAFLD in humans. Conclusion: Preclinical validation for the metabolic benefit of pharmacologically inhibiting STK25 in the context of obesity is provided. Therapeutic intervention aimed at reducing STK25 function may provide a new strategy for the treatment of patients with NAFLD, type 2 diabetes, and related complex metabolic diseases. (Hepatology Communications 2018;2:69-83).

Reduced obesity, diabetes, and steatosis upon cinnamon and grape pomace are associated with changes in gut microbiota and markers of gut barrier.

Increasing evidence suggests that polyphenols have a significant potential in the prevention and treatment of risk factors associated with metabolic syndrome. The objective of this study was to assess the metabolic outcomes of two polyphenol-containing extracts from cinnamon bark (CBE) and grape pomace (GPE) on C57BL/6J mice fed a high-fat diet (HFD) for 8 wk. Both CBE and GPE were able to decrease fat mass gain and adipose tissue inflammation in mice fed a HFD without reducing food intake. This was associated with reduced liver steatosis and lower plasma nonesterified fatty acid levels. We also observed a beneficial effect on glucose homeostasis, as evidenced by an improved glucose tolerance and a lower insulin resistance index. These ameliorations of the overall metabolic profile were associated with a significant impact on the microbial composition, which was more profound for the GPE than for the CBE. At the genus level, Peptococcus were decreased in the CBE group. In the GPE-treated group, several key genera that have been previously found to be linked with HFD, metabolic effects, and gut barrier integrity were affected: we observed a decrease of Desulfovibrio, Lactococcus, whereas Allobaculum and Roseburia were increased. In addition, the expression of several antimicrobial peptides and tight junction proteins was increased in response to both CBE and GPE supplementation, indicating an improvement of the gut barrier function. Collectively, these data suggest that CBE and GPE can ameliorate the overall metabolic profile of mice on a high-fat diet, partly by acting on the gut microbiota.

Network analyses identify liver-specific targets for treating liver diseases.

We performed integrative network analyses to identify targets that can be used for effectively treating liver diseases with minimal side effects. We first generated co-expression networks (CNs) for 46 human tissues and liver cancer to explore the functional relationships between genes and examined the overlap between functional and physical interactions. Since increased de novo lipogenesis is a characteristic of nonalcoholic fatty liver disease (NAFLD) and hepatocellular carcinoma (HCC), we investigated the liver-specific genes co-expressed with fatty acid synthase (FASN). CN analyses predicted that inhibition of these liver-specific genes decreases FASN expression. Experiments in human cancer cell lines, mouse liver samples, and primary human hepatocytes validated our predictions by demonstrating functional relationships between these liver genes, and showing that their inhibition decreases cell growth and liver fat content. In conclusion, we identified liver-specific genes linked to NAFLD pathogenesis, such as pyruvate kinase liver and red blood cell (PKLR), or to HCC pathogenesis, such as PKLR, patatin-like phospholipase domain containing 3 (PNPLA3), and proprotein convertase subtilisin/kexin type 9 (PCSK9), all of which are potential targets for drug development.

NorUrsodeoxycholic acid ameliorates cholemic nephropathy in bile duct ligated mice.

BACKGROUND & AIMS: Severe cholestasis may cause cholemic nephropathy that can be modeled in common bile duct ligated (CBDL) mice. We aimed to explore the therapeutic efficacy and mechanisms of norursodeoxycholic acid (norUDCA) in cholemic nephropathy. METHODS: In 8-week CBDL mice fed with norUDCA (prior or post CBDL) or chow we evaluated serum urea levels, urine cytology and urinary neutrophil gelatinase associated lipocalin (uNGAL), kidney and liver tissue quantification of fibrosis by hydroxyproline content and gene chip expression looking at key genes of inflammation and fibrosis. Moreover, we comprehensively analysed bile acid profiles in liver, kidney, serum and urine samples. RESULTS: NorUDCA-fed CBDL mice had significantly lower serum urea and uNGAL levels and less severe cholemic nephropathy as demonstrated by normal urine cytology, significantly reduced tubulointerstitial nephritis, and renal fibrosis as compared to controls. NorUDCA underwent extensive metabolism to produce even more hydrophilic compounds that were significantly enriched in kidneys. CONCLUSION: NorUDCA ameliorates cholemic nephropathy due to the formation of highly hydrophilic metabolites enriched in kidney. Consequently, norUDCA may represent a medical treatment for cholemic nephropathy. LAY SUMMARY: The term cholemic nephropathy describes renal dysfunction together with characteristic morphological alterations of the kidney in obstructive cholestasis that can be mimicked by ligation of the common bile duct in mice. Feeding the hydrophilic bile acid norUDCA to bile duct ligated mice leads to a significant amelioration of the renal phenotype due to the formation of highly hydrophilic metabolites enriched in the kidney and may therefore represent a medical treatment for cholemic nephropathy.