Integrative analysis of liver-specific non-coding regulatory SNPs associated with the risk of coronary artery disease.

Genetic factors underlying coronary artery disease (CAD) have been widely studied using genome-wide association studies (GWASs). However, the functional understanding of the CAD loci has been limited by the fact that a majority of GWAS variants are located within non-coding regions with no functional role. High cholesterol and dysregulation of the liver metabolism such as non-alcoholic fatty liver disease confer an increased risk of CAD. Here, we studied the function of non-coding single-nucleotide polymorphisms in CAD GWAS loci located within liver-specific enhancer elements by identifying their potential target genes using liver cis-eQTL analysis and promoter Capture Hi-C in HepG2 cells. Altogether, 734 target genes were identified of which 121 exhibited correlations to liver-related traits. To identify potentially causal regulatory SNPs, the allele-specific enhancer activity was analyzed by (1) sequence-based computational predictions, (2) quantification of allele-specific transcription factor binding, and (3) STARR-seq massively parallel reporter assay. Altogether, our analysis identified 1,277 unique SNPs that display allele-specific regulatory activity. Among these, susceptibility enhancers near important cholesterol homeostasis genes (APOB, APOC1, APOE, and LIPA) were identified, suggesting that altered gene regulatory activity could represent another way by which genetic variation regulates serum lipoprotein levels. Using CRISPR-based perturbation, we demonstrate how the deletion/activation of a single enhancer leads to changes in the expression of many target genes located in a shared chromatin interaction domain. Our integrative genomics approach represents a comprehensive effort in identifying putative causal regulatory regions and target genes that could predispose to clinical manifestation of CAD by affecting liver function.

Increased secretion of adipocyte-derived extracellular vesicles is associated with adipose tissue inflammation and the mobilization of excess lipid in human obesity.

BACKGROUND: Obesity is a worldwide epidemic characterized by adipose tissue (AT) inflammation. AT is also a source of extracellular vesicles (EVs) that have recently been implicated in disorders related to metabolic syndrome. However, our understanding of mechanistic aspect of obesity's impact on EV secretion from human AT remains limited. METHODS: We investigated EVs from human Simpson Golabi Behmel Syndrome (SGBS) adipocytes, and from AT as well as plasma of subjects undergoing bariatric surgery. SGBS cells were treated with TNFα, palmitic acid, and eicosapentaenoic acid. Various analyses, including nanoparticle tracking analysis, electron microscopy, high-resolution confocal microscopy, and gas chromatography-mass spectrometry, were utilized to study EVs. Plasma EVs were analyzed with imaging flow cytometry. RESULTS: EVs from mature SGBS cells differed significantly in size and quantity compared to preadipocytes, disagreeing with previous findings in mouse adipocytes and indicating that adipogenesis promotes EV secretion in human adipocytes. Inflammatory stimuli also induced EV secretion, and altered EV fatty acid (FA) profiles more than those of cells, suggesting the role of EVs as rapid responders to metabolic shifts. Visceral AT (VAT) exhibited higher EV secretion compared to subcutaneous AT (SAT), with VAT EV counts positively correlating with plasma triacylglycerol (TAG) levels. Notably, the plasma EVs of subjects with obesity contained a higher number of adiponectin-positive EVs than those of lean subjects, further demonstrating higher AT EV secretion in obesity. Moreover, plasma EV counts of people with obesity positively correlated with body mass index and TNF expression in SAT, connecting increased EV secretion with AT expansion and inflammation. Finally, EVs from SGBS adipocytes and AT contained TAGs, and EV secretion increased despite signs of less active lipolytic pathways, indicating that AT EVs could be involved in the mobilization of excess lipids into circulation. CONCLUSIONS: We are the first to provide detailed FA profiles of human AT EVs. We report that AT EV secretion increases in human obesity, implicating their role in TAG transport and association with adverse metabolic parameters, thereby emphasizing their role in metabolic disorders. These findings promote our understanding of the roles that EVs play in human AT biology and metabolic disorders.

Effectiveness of workplace choice architecture modification for healthy eating and daily physical activity.

BACKGROUND: Modifying the choice architecture of behavioural contexts can facilitate health behaviour change, but existing evidence builds mostly on small-scale interventions limited in duration, targets, strategies, and settings. We evaluated the effectiveness of a one-year hybrid type 2 implementation-effectiveness trial aimed at promoting healthy eating and daily physical activity with subtle modifications to the choice architecture of heterogeneous worksites. The intervention was contextualised to and integrated into the routine operations of each worksite. Effectiveness was evaluated in a quasi-experimental pre-post design. METHODS: Intervention sites (n = 21) implemented a median of two (range 1-9) intervention strategies for healthy eating and one (range 1-5) for physical activity. Questionnaires pre (n = 1126) and post (n = 943) intervention surveyed employees' behavioural patterns at work (food consumption: vegetables/roots, fruit/berries, nuts/almonds/seeds, sweet treats, fast food, water; physical activity: restorative movement, exercise equipment use, stair use). The post-intervention questionnaire also measured employees' perception of and response to three intervention strategies: a packed lunch recipe campaign, a fruit crew-strategy, and movement prompts. Multi- and single-level regression models evaluated effectiveness, treating intervention as a continuous predictor formed of the site-specific dose (n intervention strategies employed) and mean quality (three-point rating per strategy halfway and at the end of the intervention) of implementation relevant to each outcome. RESULTS: Multinomial logistic regression models found the intervention significantly associated with a favourable change in employees' fruit and berry consumption (interaction effect of time and implementation p = 0.006) and with an unfavourable change in sweet treat consumption (p = 0.048). The evidence was strongest for the finding concerning fruit/berry consumption-an outcome that sites with greater dose and quality of implementation targeted by using strategies that reduced the physical effort required to have fruit/berries at work and by covering multiple eating-related contexts at the worksite. The quality of implementation was positively associated with the perception of (p = 0.044) and response to (p = 0.017) the packed lunch recipes, and with response to the fruit crew-strategy (p < 0.001). CONCLUSIONS: The results suggest that a contextualised, multicomponent choice architecture intervention can positively influence eating behaviour in diverse real-world settings over a one-year period, and that higher implementation quality can enhance intervention perception and response. However, outcomes may depend on the type of intervention strategies used and the extent of their delivery.

Programmed cell death 1 genetic variant and liver damage in nonalcoholic fatty liver disease.

BACKGROUND AND AIMS: Programmed cell death 1/programmed cell death-ligand 1 (PD-1/PDL-1) axis has been reported to modulate liver inflammation and progression to hepatocellular carcinoma (HCC) in patients with nonalcoholic fatty liver disease (NAFLD). Here, we examined whether the PDCD1 variation is associated with NAFLD severity in individuals with liver biopsy. METHODS: We examined the impact of PDCD1 gene variants on HCC, as robust severe liver disease phenotype in UK Biobank participants. The strongest genetic association with the rs13023138 G>C variation was subsequently tested for association with liver damage in 2889 individuals who underwent liver biopsy for suspected nonalcoholic steatohepatitis (NASH). Hepatic transcriptome was examined by RNA-Seq in a subset of NAFLD individuals (n = 121). Transcriptomic and deconvolution analyses were performed to identify biological pathways modulated by the risk allele. RESULTS: The rs13023138 C>G showed the most robust association with HCC in UK Biobank (p = 5.28E-4, OR = 1.32, 95% CI [1.1, 1.5]). In the liver biopsy cohort, rs13023138 G allele was independently associated with severe steatosis (OR 1.17, 95% CI 1.02-1.34; p = .01), NASH (OR 1.22, 95% CI 1.09-1.37; p < .001) and advanced fibrosis (OR 1.26, 95% CI 1.06-1.50; p = .007). At deconvolution analysis, rs13023138 G>C allele was linked to higher hepatic representation of M1 macrophages, paralleled by upregulation of pathways related to inflammation and higher expression of CXCR6. CONCLUSIONS: The PDCD1 rs13023138 G allele was associated with HCC development in the general population and with liver disease severity in patients at high risk of NASH.

Immigrants' perspectives on healthy life and healthy lifestyle counseling: a focus group study.

BACKGROUND: Immigrants have a higher risk of developing chronic diseases than the general population, but there is a lack of knowledge about appropriate counseling models to promote their health. This study aimed to explore Asian and Russian immigrants' perspectives in Finland on healthy lifestyle and healthy lifestyle counseling to improve the quality of the counseling in primary health care services to prevent type 2 diabetes and other chronic diseases. METHODS: We conducted a qualitative study using semi-structured questions for eight focus groups. The participants were 46 adult immigrants (21 Asian and 25 Russian) living legally in Finland. Interviews were transcribed verbatim, coded, and analyzed using deductive content analysis. RESULTS: We identified three themes regarding healthy lifestyle: (1) limited knowledge on different dimensions of healthy lifestyle, (2) understanding the impact of culture and community on healthy living, and (3) changing the lifestyle to live healthier after immigration. Moreover, we found three themes regarding healthy lifestyle counseling: (1) shortcomings in health care for providing healthy lifestyle counseling, such as lack of educational materials and miscommunication, (2) influential individual factors for using healthy lifestyle counseling, such as stress, and (3) positive outcomes of healthy lifestyle counseling. CONCLUSION: Developing a culturally tailored healthy lifestyle counseling program for the enhancement of immigrants' knowledge regarding healthy lifestyle with consideration of cultural and linguistic aspects is recommended for preventing chronic diseases among immigrants.

Acceptability of workplace choice architecture modification for healthy behaviours.

BACKGROUND: Altering the choice architecture of decision contexts can assist behaviour change, but the acceptability of this approach has sparked debate. Considering hypothetical interventions, people generally welcome the approach for promoting health, but little evidence exists on acceptance in the real world. Furthermore, research has yet to explore the implementers' perspective, acknowledging the multidimensionality of the acceptability construct. Addressing these knowledge gaps, this study evaluated the acceptability of a quasi-experimental implementation-effectiveness trial that modified the worksite choice architecture for healthy eating and daily physical activity. METHODS: Fifty-three worksites participated in the 12-month intervention and implemented altogether 23 choice architecture strategies (Mdn 3/site), including point-of-choice prompts and changes to choice availability or accessibility. Retrospective acceptability evaluation built on deductive qualitative content analysis of implementer interviews (n = 65) and quantitative analysis of an employee questionnaire (n = 1124). Qualitative analysis examined implementers' thoughts and observations of the intervention and its implementation, considering six domains of the Theoretical Framework of Acceptability: ethicality, affective attitude, burden, intervention coherence, opportunity costs, and perceived effectiveness. Quantitative analysis examined employees' acceptance (7-point Likert scale) of eight specific intervention strategies using Friedman test and mixed-effects logistic regression. RESULTS: Implementers considered the choice architecture approach ethical for workplace health promotion, reported mostly positive affective attitudes to and little burden because of the intervention. Intervention coherence supported acceptance through increased interest in implementation, whereas low perceived utility and high intensity of implementation reduced cost acceptance. Perceived effectiveness was mixed and varied along factors related to the implementer, social/physical work environment, employer, and employee. Employees showed overall high acceptance of evaluated strategies (Mdn 7, IQR 6.4-7), though strategies replacing unhealthy foods with healthier alternatives appeared less supported than providing information or enhancing healthy option availability or accessibility (p-values < 0.02). Greater proportion of male employees per site predicted lower overall acceptance (OR 4.4, 95% CI 1.2-16.5). CONCLUSIONS: Work communities appear to approve workplace choice architecture interventions for healthy eating and physical activity, but numerous factors influence acceptance and warrant consideration in future interventions. The study contributes with a theory-based, multidimensional evaluation that considered the perspectives of implementers and influenced individuals across heterogeneous real-world settings.

High estradiol levels during a long agonist IVF protocol are associated with decreased food intake, higher leptin concentrations, and lower levels of high-sensitivity C-reactive protein.

PURPOSE: To study whether different hormonal phases affect appetite regulation, food intake, and concentrations of leptin, glucagon-like peptide-1 (GLP-1), and high-sensitivity C-reactive protein (hs-CRP) during a long agonist in vitro fertilization (IVF) protocol. METHODS: Fifty-four infertile women were encountered thrice, the first of which was at the beginning of their period (low estradiol). The other two visits were during a gonadotrophin-releasing hormone (GnRH) analog downregulation (low estradiol) and at the end of a follicle-stimulating hormone (FSH) stimulation (high estradiol). The first visit was the reference; the women served as their controls. The concentrations of leptin, GLP-1, and hs-CRP were assessed from plasma. Dietary intake was assessed using food records (FRs). In addition, weight, height, body mass index (BMI), and plasma levels of estradiol, glucose, HbA1c, insulin, and lipids were monitored. Twenty-six of the subjects also had a postprandial test. RESULTS: During the stimulation protocol, leptin concentrations elevated (P < 0.001), and energy intake decreased (P = 0.03), while estradiol levels increased (P < 0.001). GLP-1 levels unchanged (P = 0.75) and hs-CRP (P = 0.03) concentrations diminished, while estradiol levels increased. CONCLUSION: No increased food intake or weight gain occurred during the stimulation protocol; thus, leptin may protect from overeating during high estradiol levels, and leptin resistance may not occur during a short follow-up. Also, a favorable anti-inflammatory effect was detected. During this study, we observed no harmful metabolic effects, which might affect negatively maternal health.

Distinct contributions of metabolic dysfunction and genetic risk factors in the pathogenesis of non-alcoholic fatty liver disease.

BACKGROUND & AIMS: There is substantial inter-individual variability in the risk of non-alcoholic fatty liver disease (NAFLD). Part of which is explained by insulin resistance (IR) ('MetComp') and part by common modifiers of genetic risk ('GenComp'). We examined how IR on the one hand and genetic risk on the other contribute to the pathogenesis of NAFLD. METHODS: We studied 846 individuals: 492 were obese patients with liver histology and 354 were individuals who underwent intrahepatic triglyceride measurement by proton magnetic resonance spectroscopy. A genetic risk score was calculated using the number of risk alleles in PNPLA3, TM6SF2, MBOAT7, HSD17B13 and MARC1. Substrate concentrations were assessed by serum NMR metabolomics. In subsets of participants, non-esterified fatty acids (NEFAs) and their flux were assessed by D5-glycerol and hyperinsulinemic-euglycemic clamp (n = 41), and hepatic de novo lipogenesis (DNL) was measured by D2O (n = 61). RESULTS: We found that substrate surplus (increased concentrations of 28 serum metabolites including glucose, glycolytic intermediates, and amino acids; increased NEFAs and their flux; increased DNL) characterized the 'MetComp'. In contrast, the 'GenComp' was not accompanied by any substrate excess but was characterized by an increased hepatic mitochondrial redox state, as determined by serum ß-hydroxybutyrate/acetoacetate ratio, and inhibition of hepatic pathways dependent on tricarboxylic acid cycle activity, such as DNL. Serum ß-hydroxybutyrate/acetoacetate ratio correlated strongly with all histological features of NAFLD. IR and hepatic mitochondrial redox state conferred additive increases in histological features of NAFLD. CONCLUSIONS: These data show that the mechanisms underlying 'Metabolic' and 'Genetic' components of NAFLD are fundamentally different. These findings may have implications with respect to the diagnosis and treatment of NAFLD. LAY SUMMARY: The pathogenesis of non-alcoholic fatty liver disease can be explained in part by a metabolic component, including obesity, and in part by a genetic component. Herein, we demonstrate that the mechanisms underlying these components are fundamentally different: the metabolic component is characterized by hepatic oversupply of substrates, such as sugars, lipids and amino acids. In contrast, the genetic component is characterized by impaired hepatic mitochondrial function, making the liver less able to metabolize these substrates.

Rare ATG7 genetic variants predispose patients to severe fatty liver disease.

BACKGROUND & AIMS: Non-alcoholic fatty liver disease (NAFLD) is the leading cause of liver disorders and has a strong heritable component. The aim of this study was to identify new loci that contribute to severe NAFLD by examining rare variants. METHODS: We performed whole-exome sequencing in individuals with NAFLD and advanced fibrosis or hepatocellular carcinoma (n = 301) and examined the enrichment of likely pathogenic rare variants vs. the general population. This was followed by validation at the gene level. RESULTS: In patients with severe NAFLD, we observed an enrichment of the p.P426L variant (rs143545741 C>T; odds ratio [OR] 5.26, 95% CI 2.1-12.6; p = 0.003) of autophagy-related 7 (ATG7), which we characterized as a loss-of-function, vs. the general population, and an enrichment in rare variants affecting the catalytic domain (OR 13.9; 95% CI 1.9-612; p = 0.002). In the UK Biobank cohort, loss-of-function ATG7 variants increased the risk of cirrhosis and hepatocellular carcinoma (OR 3.30; 95% CI 1.1-7.5 and OR 12.30, 95% CI 2.6-36, respectively; p <0.001 for both). The low-frequency loss-of-function p.V471A variant (rs36117895 T>C) was also associated with severe NAFLD in the clinical cohort (OR 1.7; 95% CI 1.2-2.5; p = 0.003), predisposed to hepatocellular ballooning (p = 0.007) evolving to fibrosis in the Liver biopsy cohort (n = 2,268), and was associated with liver injury in the UK Biobank (aspartate aminotransferase levels, p <0.001), with a larger effect in severely obese individuals in whom it was linked to hepatocellular carcinoma (p = 0.009). ATG7 protein localized to periportal hepatocytes, particularly in the presence of ballooning. In the Liver Transcriptomic cohort (n = 125), ATG7 expression correlated with suppression of the TNFα pathway, which was conversely upregulated in p.V471A carriers. CONCLUSIONS: We identified rare and low-frequency ATG7 loss-of-function variants that promote NAFLD progression by impairing autophagy and facilitating ballooning and inflammation. LAY SUMMARY: We found that rare mutations in a gene called autophagy-related 7 (ATG7) increase the risk of developing severe liver disease in individuals with dysmetabolism. These mutations cause an alteration in protein function and impairment of self-renewal of cellular content, leading to liver damage and inflammation.

Exome-Wide Association Study on Alanine Aminotransferase Identifies Sequence Variants in the GPAM and APOE Associated With Fatty Liver Disease.

BACKGROUND & AIMS: Fatty liver disease (FLD) is a growing epidemic that is expected to be the leading cause of end-stage liver disease within the next decade. Both environmental and genetic factors contribute to the susceptibility of FLD. Several genetic variants contributing to FLD have been identified in exome-wide association studies. However, there is still a missing hereditability indicating that other genetic variants are yet to be discovered. METHODS: To find genes involved in FLD, we first examined the association of missense and nonsense variants with alanine aminotransferase at an exome-wide level in 425,671 participants from the UK Biobank. We then validated genetic variants with liver fat content in 8930 participants in whom liver fat measurement was available, and replicated 2 genetic variants in 3 independent cohorts comprising 2621 individuals with available liver biopsy. RESULTS: We identified 190 genetic variants independently associated with alanine aminotransferase after correcting for multiple testing with Bonferroni method. The majority of these variants were not previously associated with this trait. Among those associated, there was a striking enrichment of genetic variants influencing lipid metabolism. We identified the variants rs2792751 in GPAM/GPAT1, the gene encoding glycerol-3-phosphate acyltransferase, mitochondrial, and rs429358 in APOE, the gene encoding apolipoprotein E, as robustly associated with liver fat content and liver disease after adjusting for multiple testing. Both genes affect lipid metabolism in the liver. CONCLUSIONS: We identified 2 novel genetic variants in GPAM and APOE that are robustly associated with steatosis and liver damage. These findings may help to better elucidate the genetic susceptibility to FLD onset and progression.

FADS1 rs174550 genotype and high linoleic acid diet modify plasma PUFA phospholipids in a dietary intervention study.

INTRODUCTION: Fatty acid desaturase 1 (FADS1) gene encodes for delta-5 desaturase enzyme which is needed in conversion of linoleic acid (LA) to arachidonic acid (AA). Recent studies have shown that response to dietary PUFAs differs between the genotypes in circulating fatty acids. However, interactions between the FADS1 genotype and dietary LA on overall metabolism have not been studied. OBJECTIVES: We aimed to examine the interactions of FADS1 rs174550 genotypes (TT and CC) and high-LA diet to identify plasma metabolites that respond differentially to dietary LA according to the FADS1 genotype. METHODS: A total of 59 men (TT n = 26, CC n = 33) consumed a sunflower oil supplemented diet for 4 weeks. Daily dose of 30, 40, or 50 ml was calculated based on body mass index. It resulted in 17-28 g of LA on top of the usual daily intake. Fasting plasma samples at the beginning and at the end of the intervention were analyzed with LC-MS/MS non-targeted metabolomics method. RESULTS: At the baseline, the carriers of FADS1 rs174550-TT genotype had higher abundance of long-chain PUFA phospholipids compared to the FADS1 rs174550-CC one. In response to the high-LA diet, LA phospholipids and long-chain acylcarnitines increased and lysophospholipids decreased in fasting plasma similarly in both genotypes. LysoPE (20:4), LysoPC (20:4), and PC (16:0_20:4) decreased and cortisol increased in the carriers of rs174550-CC genotype; however, these genotype-diet interactions were not significant after correction for multiple testing. CONCLUSION: Our findings show that both FADS1 rs174550 genotype and high-LA diet modify plasma phospholipid composition. TRIAL REGISTRATION: The study was registered to ClinicalTrials: NCT02543216, September 7, 2015 (retrospectively registered).

The FADS1 genotypes modify the effect of linoleic acid-enriched diet on adipose tissue inflammation via pro-inflammatory eicosanoid metabolism.

PURPOSE: Fatty acid desaturase (FADS) variants associate with fatty acid (FA) and adipose tissue (AT) metabolism and inflammation. Thus, the role of FADS1 variants in the regulation of dietary linoleic acid (LA)-induced effects on AT inflammation was investigated. METHODS: Subjects homozygotes for the TT and CC genotypes of the FADS1-rs174550 (TT, n = 25 and CC, n = 28) or -rs174547 (TT, n = 42 and CC, n = 28), were either recruited from the METabolic Syndrome In Men cohort to participate in an intervention with LA-enriched diet (FADSDIET) or from the Kuopio Obesity Surgery (KOBS) study. GC and LC-MS for plasma FA proportions and eicosanoid concentrations and AT gene expression for AT inflammatory score (AT-InSc) was determined. RESULTS: We observed a diet-genotype interaction between LA-enriched diet and AT-InSc in the FADSDIET. In the KOBS study, interleukin (IL)1 beta mRNA expression in AT was increased in subjects with the TT genotype and highest LA proportion. In the FADSDIET, n-6/LA proportions correlated positively with AT-InSc in those with the TT genotype but not with the CC genotype after LA-enriched diet. Specifically, LA- and AA-derived pro-inflammatory eicosanoids related to CYP450/sEH-pathways correlated positively with AT-InSc in those with the TT genotype, whereas in those with the CC genotype, the negative correlations between pro-inflammatory eicosanoids and AT-InSc related to COX/LOX-pathways. CONCLUSIONS: LA-enriched diet increases inflammatory AT gene expression in subjects with the TT genotype, while CC genotype could play a protective role against LA-induced AT inflammation. Overall, the FADS1 variant could modify the dietary LA-induced effects on AT inflammation through the differential biosynthesis of AA-derived eicosanoids.

Digitally Supported Lifestyle Intervention to Prevent Type 2 Diabetes Through Healthy Habits: Secondary Analysis of Long-Term User Engagement Trajectories in a Randomized Controlled Trial.

BACKGROUND: Digital health interventions may offer a scalable way to prevent type 2 diabetes (T2D) with minimal burden on health care systems by providing early support for healthy behaviors among adults at increased risk for T2D. However, ensuring continued engagement with digital solutions is a challenge impacting the expected effectiveness. OBJECTIVE: We aimed to investigate the longitudinal usage patterns of a digital healthy habit formation intervention, BitHabit, and the associations with changes in T2D risk factors. METHODS: This is a secondary analysis of the StopDia (Stop Diabetes) study, an unblinded parallel 1-year randomized controlled trial evaluating the effectiveness of the BitHabit app alone or together with face-to-face group coaching in comparison with routine care in Finland in 2017-2019 among community-dwelling adults (aged 18 to 74 years) at an increased risk of T2D. We used longitudinal data on usage from 1926 participants randomized to the digital intervention arms. Latent class growth models were applied to identify user engagement trajectories with the app during the study. Predictors for trajectory membership were examined with multinomial logistic regression models. Analysis of covariance was used to investigate the association between trajectories and 12-month changes in T2D risk factors. RESULTS: More than half (1022/1926, 53.1%) of the participants continued to use the app throughout the 12-month intervention. The following 4 user engagement trajectories were identified: terminated usage (904/1926, 46.9%), weekly usage (731/1926, 38.0%), twice weekly usage (208/1926, 10.8%), and daily usage (83/1926, 4.3%). Active app use during the first month, higher net promoter score after the first 1 to 2 months of use, older age, and better quality of diet at baseline increased the odds of belonging to the continued usage trajectories. Compared with other trajectories, daily usage was associated with a higher increase in diet quality and a more pronounced decrease in BMI and waist circumference at 12 months. CONCLUSIONS: Distinct long-term usage trajectories of the BitHabit app were identified, and individual predictors for belonging to different trajectory groups were found. These findings highlight the need for being able to identify individuals likely to disengage from interventions early on, and could be used to inform the development of future adaptive interventions. TRIAL REGISTRATION: ClinicalTrials.gov NCT03156478; https://clinicaltrials.gov/ct2/show/NCT03156478. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): RR2-10.1186/s12889-019-6574-y.

LPIAT1/MBOAT7 depletion increases triglyceride synthesis fueled by high phosphatidylinositol turnover.

OBJECTIVE: Non-alcoholic fatty liver disease (NAFLD) is a common prelude to cirrhosis and hepatocellular carcinoma. The genetic rs641738 C>T variant in the lysophosphatidylinositol acyltransferase 1 (LPIAT1)/membrane bound O-acyltransferase domain-containing 7, which incorporates arachidonic acid into phosphatidylinositol (PI), is associated with the entire spectrum of NAFLD. In this study, we investigated the mechanism underlying this association in mice and cultured human hepatocytes. DESIGN: We generated the hepatocyte-specific Lpiat1 knockout mice to investigate the function of Lpiat1 in vivo. We also depleted LPIAT1 in cultured human hepatic cells using CRISPR-Cas9 systems or siRNA. The effect of LPIAT1-depletion on liver fibrosis was examined in mice fed high fat diet and in liver spheroids. Lipid species were measured using liquid chromatography-electrospray ionisation mass spectrometry. Lipid metabolism was analysed using radiolabeled glycerol or fatty acids. RESULTS: The hepatocyte-specific Lpiat1 knockout mice developed hepatic steatosis spontaneously, and hepatic fibrosis on high fat diet feeding. Depletion of LPIAT1 in cultured hepatic cells and in spheroids caused triglyceride accumulation and collagen deposition. The increase in hepatocyte fat content was due to a higher triglyceride synthesis fueled by a non-canonical pathway. Indeed, reduction in the PI acyl chain remodelling caused a high PI turnover, by stimulating at the same time PI synthesis and breakdown. The degradation of PI was mediated by a phospholipase C, which produces diacylglycerol, a precursor of triglyceride. CONCLUSION: We found a novel pathway fueling triglyceride synthesis in hepatocytes, by a direct metabolic flow of PI into triglycerides. Our findings provide an insight into the pathogenesis and therapeutics of NAFLD.

Serum aromatic and branched-chain amino acids associated with NASH demonstrate divergent associations with serum lipids.

BACKGROUND & AIMS: Non-alcoholic fatty liver disease (NAFLD) has been associated with multiple metabolic abnormalities. By applying a non-targeted metabolomics approach, we aimed at investigating whether serum metabolite profile that associates with NAFLD would differ in its association with NAFLD-related metabolic risk factors. METHODS & RESULTS: A total of 233 subjects (mean ± SD: 48.3 ± 9.3 years old; BMI: 43.1 ± 5.4 kg/m2 ; 64 male) undergoing bariatric surgery were studied. Of these participants, 164 with liver histology could be classified as normal liver (n = 79), simple steatosis (SS, n = 40) or non-alcoholic steatohepatitis (NASH, n = 45). Among the identified fasting serum metabolites with higher levels in those with NASH when compared to those with normal phenotype were the aromatic amino acids (AAAs: tryptophan, tyrosine and phenylalanine), the branched-chain amino acids (BCAAs: leucine and isoleucine), a phosphatidylcholine (PC(16:0/16:1)) and uridine (all FDRp < 0.05). Only tryptophan was significantly higher in those with NASH compared to those with SS (FDRp < 0.05). Only the AAAs tryptophan and tyrosine correlated positively with serum total and LDL cholesterol (FDRp < 0.1), and accordingly, with liver LDLR at mRNA expression level. In addition, tryptophan was the single AA associated with liver DNA methylation of CpG sites known to be differentially methylated in those with NASH. CONCLUSIONS: We found that serum levels of the NASH-related AAAs and BCAAs demonstrate divergent associations with serum lipids. The specific correlation of tryptophan with LDL-c may result from the molecular events affecting LDLR mRNA expression and NASH-associated methylation of genes in the liver.

NR1H4 rs35724 G>C variant modulates liver damage in nonalcoholic fatty liver disease.

BACKGROUND AND AIMS: Farnesoid X receptor (FXR) plays a key role in bile acid and lipid homeostasis. Experimental evidence suggests that it can modulate liver damage related to nonalcoholic fatty liver disease (NAFLD). We examined the impact of the NR1H4 rs35724 G>C, encoding for FXR, on liver damage in a large cohort of patients at risk of steatohepatitis. METHODS: We considered 2,660 consecutive individuals at risk of steatohepatitis with liver histology. The rs35724 G>C polymorphisms were genotyped by TaqMan assays. Gene expression was evaluated by RNASeq in a subset of patients (n = 124). RESULTS: The NR1H4 rs35724 CC genotype, after adjusting for clinic-metabolic and genetic confounders and for enrolling centre, was protective against severity of steatosis (GG vs CC OR 0.77, 95% CI 0.62-0.95; P = .01), steatohepatitis (GG vs CC OR 0.62, 95% CI 0.47-0.83; P = .001) and severity of fibrosis (GG vs CC OR 0.83, 95% CI 0.67-0.98; P = .04). The C allele was associated with higher total circulating cholesterol (P = .01). Patients carrying the NR1H4 rs35724 C allele had significantly higher hepatic mRNA levels of FXR and were associated with higher hepatic FGFR4 and Cyp39A1 that are in turn involved in bile acid synthesis. CONCLUSIONS: Increased hepatic FXR expression due to the NR1H4 rs35724 C allele is linked to higher serum cholesterol but protects against steatosis, steatohepatitis and liver fibrosis. The translational relevance of these results for patient risk stratification and FXR-targeted therapy warrants further investigation.

PCSK9 rs11591147 R46L loss-of-function variant protects against liver damage in individuals with NAFLD.

BACKGROUND AND AIMS: The proprotein convertase subtilisin/kexin type 9 (PCSK9) plays a key role in cholesterol homeostasis, and its inhibition represents an effective therapy to lower low-density lipoprotein cholesterol (LDL-C) levels. In this study, we examined the impact of the PCSK9 rs11591147 loss-of-function (LOF) variant on liver damage in a multicenter collection of patients at risk of nonalcoholic steatohepatitis (NASH), in clinical samples and experimental models. METHODS: We considered 1874 consecutive individuals at risk of NASH as determined by histology. The SNP rs11591147, encoding for the p.R46L variant of PCSK9, was genotyped by TaqMan assays. We also evaluated 1) PCSK9 mRNA hepatic expression in human liver, and 2) the impact of a NASH-inducing diet in mice with hepatic overexpression of human PCSK9. RESULTS: Carriers of PCSK9 rs11591147 had lower circulating LDL-C levels and were protected against nonalcoholic fatty liver disease (NAFLD) (OR: 0.42; 95% CI: 0.22-0.81; P = .01), NASH (OR: 0.48; 95% CI: 0.26-0.87; P = .01) and more severe fibrosis (OR: 0.55; 95% CI: 0.32-0.94; P = .03) independently of clinical, metabolic and genetic confounding factors. PCSK9 hepatic expression was directly correlated with liver steatosis (P = .03). Finally, liver-specific overexpression of human PCSK9 in male mice drives NAFLD and fibrosis upon a dietary challenge. CONCLUSIONS: In individuals at risk of NASH, PCSK9 was induced with hepatic fat accumulation and PCSK9 rs11591147 LOF variant was protective against liver steatosis, NASH and fibrosis, suggesting that PCSK9 inhibition may be a new therapeutic strategy to treat NASH.

Mucosal-associated invariant T cell alterations during the development of human type 1 diabetes.

AIMS/HYPOTHESIS: Mucosal-associated invariant T (MAIT) cells are innate-like T cells that recognise derivatives of bacterial riboflavin metabolites presented by MHC-Ib-related protein 1 (MR1) molecules and are important effector cells for mucosal immunity. Their development can be influenced by the intestinal microbiome. Since the development of type 1 diabetes has been associated with changes in the gut microbiome, this can be hypothesised to lead to alterations in circulating MAIT cells. Accordingly, peripheral blood MAIT cell alterations have been reported previously in patients with type 1 diabetes. However, a comprehensive analysis of the frequency and phenotype of circulating MAIT cells at different stages of type 1 diabetes progression is currently lacking. METHODS: We analysed the frequency, phenotype and functionality of peripheral blood MAIT cells, as well as γδ T cells, invariant natural killer T (iNKT) cells and natural killer (NK) cells with flow cytometry in a cross-sectional paediatric cohort (aged 2-15) consisting of 51 children with newly diagnosed type 1 diabetes, 27 autoantibody-positive (AAb+) at-risk children, and 113 healthy control children of similar age and HLA class II background. The frequency of MAIT cells was also assessed in a separate cross-sectional adult cohort (aged 19-39) of 33 adults with established type 1 diabetes and 37 healthy individuals of similar age. RESULTS: Children with newly diagnosed type 1 diabetes displayed a proportional increase of CD8-CD27- MAIT cells compared with healthy control children (median 4.6% vs 3.1% of MAIT cells, respectively, p = 0.004), which was associated with reduced expression of C-C chemokine receptor (CCR)5 (median 90.0% vs 94.3% of MAIT cells, p = 0.02) and ß7 integrin (median 73.5% vs 81.7% of MAIT cells, p = 0.004), as well as decreased production of IFN-γ (median 57.1% vs 69.3% of MAIT cells, p = 0.04) by the MAIT cells. The frequency of MAIT cells was also decreased in AAb+ children who later progressed to type 1 diabetes compared with healthy control children (median 0.44% vs 0.96% of CD3+ T cells, p = 0.04), as well as in adult patients with a short duration of type 1 diabetes (less than 6 years after diagnosis) compared with control individuals (median 0.87% vs 2.19% of CD3+ T cells, p = 0.007). No alterations in γδ T cell, iNKT cell or NK cell frequencies were observed in children with type 1 diabetes or in AAb+ children, with the exception of an increased frequency of IL-17A+ γδ T cells in children with newly diagnosed diabetes compared with healthy control children (median 1.58% vs 1.09% of γδ T cells, p = 0.002). CONCLUSIONS/INTERPRETATION: Changes in the frequency and phenotype of circulating MAIT cells were detectable before, at the onset and after diagnosis of type 1 diabetes in cross-sectional cohorts. Our results suggest a possible temporal association between peripheral blood MAIT cell alterations and the clinical onset of type 1 diabetes. Graphical abstract.

The effect of different estradiol levels on carotid artery distensibility during a long agonist IVF protocol.

BACKGROUND: This study was made to figure out, does low and high estradiol levels during in vitro fertilization (IVF) cycles have a different effect on carotid artery distensibility (Cdis), carotid artery diameter (Cdia), blood pressure and metabolic factors? Can the stimulation protocol be considered safe to women's vasculature? METHODS: We studied 28 women having a long agonist protocol IVF-treatment in Kuopio University Hospital during the years 2011-2016. Patients were examined at three time points: in the beginning of their own period (low estradiol), during the gonadotrophin releasing hormone (GnRH) analogue downregulation (low estradiol) and during the follicle stimulating hormone (FSH) stimulation (high estradiol). Women served as their own controls and their menstrual phase (2- to 5-day period after the beginning of menstruation with low estrogen) was used as the reference. Cdis and Cdia were assessed using ultrasound. Blood pressure, weight, estradiol levels and lipids were monitored. RESULTS: Cdis, Cdia, systolic and diastolic blood pressures peaked during the GnRH-analogue treatment with the lowest estradiol levels. Cdis, Cdia and systolic blood pressures declined by 11% (P = 0.002), 3,8% (P < 0.001) and 2,5% (P = 0.026) during the FSH-stimulation when the estradiol levels were high. Cdis correlated significantly (P < 0.05) with systolic blood pressure, diastolic blood pressure and triglycerides in high estrogenic environment and with diastolic blood pressure (P < 0.05) when estrogen profiles were low. CONCLUSIONS: Carotid artery stiffens during the high estradiol levels compared to low levels and this was not explained by the higher diameter of the carotid artery, hyperlipidemia or blood pressure profiles. All the changes in Cdis and Cdia are variations of normal, and if there is no history of cardiovascular problems, it can be considered, that the stimulation protocol is not hazardous to vasculature.

Genetic variants in the MTHFR are not associated with fatty liver disease.

The common missense sequence variants of methylenetetrahydrofolate reductase (MTHFR), rs1801131 (c.A1298C) and rs1801133 (c.C677T), favour the development of hyperhomocysteinemia and diminished DNA methylation. Previous studies, carried out in small series and with suboptimal characterization of the hepatic phenotype, tested the association of these genetic variants with fatty liver disease (FLD), with conflicting results. Here, we assessed the association of rs1801131 and rs1801133 with hepatic phenotype in the Liver Biopsy Cross-Sectional Cohort, a large cohort (n=1375 from Italy and 411 from Finland) of European individuals with suspect FLD associated with dysmetabolism. A total of 1786 subjects were analysed by ordinal regression analyses. The rs1801131 and the rs1801133 variants were not associated with steatosis, inflammation, ballooning or fibrosis. The present study suggests that changes in folate and methionine metabolism resulting from these 2 variants are not associated with a clinically significant impact on FLD in Europeans.