Heterogeneity of Non-Alcoholic Fatty Liver Disease : Genetic and Nutritional Modulation of Hepatic Lipid Metabolism

Non-alcoholic fatty liver disease (NAFLD) affects up to a quarter of the population in the Western countries. It is tightly associated with insulin resistance and the metabolic syndrome (’Metabolic NAFLD’) and increased risk of type 2 diabetes, cardiovascular disease and advanced liver disease. Liver fat is composed of several types of lipids, such as metabolically inert triglycerides and insulin resistance-inducing ceramides and diacylglycerols. Common polymorphisms in genes such as PNPLA3, MBOAT7 and TM6SF2 increase the risk of advanced liver disease, but do not affect insulin resistance (’Genetic NAFLD’). Studies I-III were performed to investigate whether a different composition of liver fat could explain this dissociation between the risk of liver disease and insulin resistance by determining the molecular lipid composition of liver biopsy samples of 125 patients undergoing bariatric surgery. In ’Metabolic NAFLD’, the liver was enriched in triglycerides, ceramides and diacylglycerols. In ’Genetic NAFLD’, the liver was also enriched in triglycerides, but not with ceramides or diacylglycerols. This marked difference in hepatic lipid composition could explain the dissociation between the risk of liver disease and insulin resistance in subtypes of NAFLD. Liver fat can originate from dietary fat or adipose tissue lipolysis, or hepatic de novo lipogenesis (DNL). Study IV aimed to determine the effects of dietary macronutrient composition (saturated or unsaturated fat or sugar) on overfeeding-induced (1000 kcal/day) changes in liver fat content and insulin resistance in 38 subjects. Methods included magnetic resonance imaging, stable isotope infusions, hyperinsulinemic-euglycemic clamps as well as plasma lipidomics, adipose tissue transcriptomics and fecal microbiome analyses. Overfeeding of saturated fat for 3 weeks increased liver fat content more than unsaturated fat by stimulating adipose tissue lipolysis. Saturated fat also increased insulin resistance, circulating ceramides and fecal gram-negative bacteria. Overfeeding of sugar increased liver fat content by stimulating DNL. The diets had distinct effects on the adipose tissue transcriptome. In conclusion, the magnitude and mechanisms underlying overfeeding-induced changes in liver fat content and insulin resistance depend on the macronutrient composition of the diet. Overfeeding of saturated fat was metabolically more harmful for the liver as compared to unsaturated fat or sugar. In conclusion, this series of studies demonstrated that NAFLD is a heterogenous disease. Both the common genetic polymorphisms and the macronutrient composition of the diet have a marked effect on hepatic lipid metabolism and the resulting phenotype.Alkoholiin liittymätöntä rasvamaksatautia (non-alcoholic fatty liver disease, NAFLD) sairastaa noin neljäsosa länsimaiden väestöstä. Se on vahvasti yhteydessä insuliiniresistenssiin ja metaboliseen oireyhtymään (‘metabolinen NAFLD’) sekä lisääntyneeseen tyypin 2 diabeteksen, valtimotaudin sekä edenneen maksasairauden riskiin. Maksan rasva koostuu useista rasvatyypeistä, kuten aineenvaihdunnallisesti neutraaleista triglyserideistä sekä insuliiniresistenssiä aiheuttavista keramideista ja diasyyliglyseroleista. Yleiset muutokset geeneissä, kuten PNPLA3, MBOAT7 ja TM6SF2, lisäävät edenneen maksataudin riskiä, mutta eivät vaikuta insuliiniresistenssiin (‘Geneettinen NAFLD’). Osatöissä I-III tutkittiin, selittääkö maksan erilainen rasvakoostumus tämän maksatauti- ja insuliiniresistenssiriskin irtikytkeytymisen määrittämällä se 125:n laihdutusleikkauspotilaan maksan koepalasta. ‘Metabolisessa NAFLD:ssa’ maksaan oli väkevöitynyt triglyseridejä, keramideja ja diasyyliglyseroleja. ‘Geneettisessä NAFLD:ssa’ maksaan oli niinikään väkevöitynyt triglyseridejä, mutta ei diasyyliglyseroleja tai keramideja. Tämän eron maksan rasvakoostumuksessa todettiin selittävän hyvin tautiriskien irtikytkeytyminen rasvamaksataudin eri alatyypeissä. Maksan rasva voi olla peräisin ruokavalion rasvasta, rasvakudoksen pilkkoutumisesta tai rasvan uudismuodostuksesta. Osatyössä IV tutkittiin ruokavalion makroravinnekoostumuksen (tyydyttynyt tai tyydyttymätön rasva, tai sokeri) vaikutusta ylensyönnin (1000 kcal/päivä) aiheuttamaan maksan rasvapitoisuuden ja insuliiniresistenssin muutokseen 38 tutkimushenkilöllä. Tutkimusmenetelminä käytettiin magneettiresonanssikuvantamista, stabiili isotooppi-infuusioita ja hyperinsulineemista-euglykeemista clamp-menetelmää sekä plasman lipidomiikka-, rasvakudoksen transkriptomiikka- ja suoliston mikrobianalyyseja. Tyydyttyneen rasvan ylensyönti lisäsi kolmen viikon aikana maksan rasvapitoisuutta enemmän kuin tyydyttymätön rasva lisäämällä rasvakudoksen pilkkoutumista. Tyydyttynyt rasva lisäsi myös insuliiniresistenssiä, verenkierron keramideja sekä suoliston gram-negatiivisia bakteereita. Sokerin ylensyönti lisäsi maksan rasvapitoisuutta kiihdyttämällä rasvan uudismuodostusta maksassa. Ruokavalioilla oli hyvin erilaiset vaikutukset rasvakudoksen transkriptomiikkaprofiiliin. Ylensyömisen aiheuttamien vaikutusten maksan rasvapitoisuuteen ja insuliiniresistenssiin sekä niiden taustalla oleviin mekanismeihin todettiin riippuvan ruokavalion makroravinnekoostumuksesta. Tyydyttyneen rasvan ylensyönti oli haitallisempaa maksan aineenvaihdunnalle verrattuna tyydyttymättömään rasvaan tai sokeriin. Yhteenvetona väitöskirjatutkimuksessa todettiin, että alkoholiin liittymätön rasvamaksatauti on monimuotoinen sairaus. Sekä yleiset geenimuutokset että ravinnon makroravinnekoostumus vaikuttavat maksan rasva-aineenvaihduntaan ja taudin ilmiasuun

Changes over time in the Chronic Liver Disease risk score predict liver-related outcomes: longitudinal analysis of the Whitehall II study

BACKGROUND AND AIMS: The Chronic Liver Disease (CLivD) risk score was recently shown to predict future advanced liver disease in the general population. We here investigated the impact of individual CLivD-score changes over time. METHODS: Participants of both phase 3 (baseline, 1991-1994) and phase 5 (follow-up, 1997-1999) examinations of the Whitehall II study were followed for liver-related outcomes (hospitalization, cancer, death) until December 2019 through linkage with electronic healthcare registers. The CLivD score, its modifiable components (alcohol use, waist-hip ratio [WHR], diabetes, and smoking), and their individual changes were studied. RESULTS: Among 6590 adults (mean age 50 years, 30% women) with a median 21-year follow-up, there were 80 liver outcomes. A rise in the CLivD score between baseline and follow-up examinations significantly increased the risk for liver-related outcomes (adjusted hazard ratio [aHR] 1.62, 95% confidence interval [CI] 1.01-2.60), more so in subjects with baseline intermediate-high CLivD scores (HR 2.4 for a CLivD-change) compared to minimal-low CLivD scores. Adverse changes over time in alcohol use and WHR, and new-onset diabetes also predicted liver outcomes. In contrast to WHR, changes in body weight (kg) showed a U-shaped association with liver outcomes. CONCLUSIONS: A change in the CLivD score over time corresponds to a true change in the risk for liver-related outcomes, suggesting the usefulness of the CLivD score for assessing response to liver-directed lifestyle interventions. Changes in WHR predicted liver outcomes better than changes in body weight or waist circumference, independent of body mass index, supporting the WHR in assessing risk for future liver disease

Quantitative PCR provides a simple and accessible method for quantitative microbiota profiling

The use of relative abundance data from next generation sequencing (NGS) can lead to misinterpretations of microbial community structures, as the increase of one taxon leads to the concurrent decrease of the other(s) in compositional data. Although different DNA- and cell-based methods as well as statistical approaches have been developed to overcome the compositionality problem, and the biological relevance of absolute bacterial abundances has been demonstrated, the human microbiome research has not yet adopted these methods, likely due to feasibility issues. Here, we describe how quantitative PCR (qPCR) done in parallel to NGS library preparation provides an accurate estimation of absolute taxon abundances from NGS data and hence provides an attainable solution to compositionality in high-throughput microbiome analyses. The advantages and potential challenges of the method are also discussed.Peer reviewe

Dietary carbohydrates and fats in nonalcoholic fatty liver disease

This Review discusses the role of dietary fats and carbohydrates in the pathogenesis of nonalcoholic fatty liver disease (NAFLD). Studies on the dietary habits of patients with NAFLD, and the effect on liver fat accumulation of altering dietary macronutrients, are also reviewed. The global prevalence of nonalcoholic fatty liver disease (NAFLD) has dramatically increased in parallel with the epidemic of obesity. Controversy has emerged around dietary guidelines recommending low-fat-high-carbohydrate diets and the roles of dietary macronutrients in the pathogenesis of metabolic disease. In this Review, the topical questions of whether and how dietary fats and carbohydrates, including free sugars, differentially influence the accumulation of liver fat (specifically, intrahepatic triglyceride (IHTG) content) are addressed. Focusing on evidence from humans, we examine data from stable isotope studies elucidating how macronutrients regulate IHTG synthesis and disposal, alter pools of bioactive lipids and influence insulin sensitivity. In addition, we review cross-sectional studies on dietary habits of patients with NAFLD and randomized controlled trials on the effects of altering dietary macronutrients on IHTG. Perhaps surprisingly, evidence to date shows no differential effects between free sugars, with both glucose and fructose increasing IHTG in the context of excess energy. Moreover, saturated fat raises IHTG more than polyunsaturated or monounsaturated fats, with adverse effects on insulin sensitivity, which are probably mediated in part by increased ceramide synthesis. Taken together, the data support the use of diets that have a reduced content of free sugars, refined carbohydrates and saturated fat in the treatment of NAFLD.Peer reviewe

Impact of short-term overfeeding of saturated or unsaturated fat or sugars on the gut microbiota in relation to liver fat in obese and overweight adults

Backgrounds & aims: Intestinal microbiota may be causally involved in the pathogenesis of non-alcoholic fatty liver disease (NAFLD). We aimed to study the effect of short-term overfeeding on human gut microbiota in relation to baseline and overfeeding-induced liver steatosis. We also asked whether the baseline microbiota composition is associated to the overfeeding-induced increase in liver fat. Methods: In a randomized trial, 38 overweight and obese subjects were assigned to consume an excess of 1000 kcal/day of diets rich in either saturated fat, unsaturated fat, or simple sugars for 3 weeks. Fasting blood samples and H-1-MR spectroscopy were used for extensive clinical phenotyping as previously reported (PMID: 29844096). Fecal samples were collected for the analysis of the gut microbiota using 16S rRNA amplicon sequencing, imputed metagenomics and qPCR. Microbiota results were correlated with dietary intakes and clinical measurements before and during overfeeding. Results: The overall community structure of the microbiota remained highly stable and personalized during overfeeding based on between-sample Bray-Curtis dissimilarity, but the relative abundances of individual taxa were altered in a diet-specific manner: overfeeding saturated fat increased Proteobacteria, while unsaturated fat increased butyrate producers. Sugar overfeeding increased Lactococcus and Escherichia coli. Imputed functions of the gut microbiota were not affected by overfeeding. Several taxa affected by overfeeding significantly correlated with the changes in host metabolic markers. The baseline levels of proteobacterial family Desulfovibrionaceae, and especially genus Bilophila, were significantly associated to overfeeding-induced liver fat increase independently of the diet arm. In general, limited overlap was observed between the overfeeding-induced microbiota changes and the liver fat-associated microbiota features at baseline. Conclusions: Our work indicates that the human gut microbiota is resilient to short-term overfeeding on community level, but specific taxa are altered on diet composition-dependent manner. Generalizable microbiota signatures directly associated with liver steatosis could not be identified. Instead, the carriage of Bilophila was identified as a potential novel risk factor for diet-induced liver steatosis in humans. (C) 2020 Published by Elsevier Ltd.Peer reviewe

Natural Course of Nonalcoholic Fatty Liver Disease and Type 2 Diabetes in Patients With Human Immunodeficiency Virus With and Without Combination Antiretroviral Therapy-associated Lipodystrophy : A 16-Year Follow-up Study

Background: Abnormal glucose metabolism and nonalcoholic fatty liver disease (NAFLD) are common in patients with human immunodeficiency virus (HIV+ patients), but longitudinal data are lacking. We determined the natural course of NAFLD (liver fat [LFAT]) and type 2 diabetes mellitus (T2DM) in HIV+ patients with and without lipodystrophy (LD+ and LD-, respectively) during a 16-year longitudinal study. Methods: LFAT (by proton magnetic resonance spectroscopy) and clinical characteristics were measured in 41 HIV+ patients at baseline and after 16 years. Liver fibrosis was estimated by measuring liver stiffness using transient elastography (TE) and magnetic resonance elastography (MRE) at 16 years. We also longitudinally studied 28 healthy subjects. Results: During follow-up, the HIV+ patients gained more body fat (8.6% 0.7%) than the control patients (4.5% 0.6%, P <.001). Features of insulin resistance increased significantly in the HIV+ patients but not the control patients. A significant proportion (20%, P <.01 vs 0% at baseline) of the HIV+ but none of the control patients developed T2DM. LFAT was significantly higher at baseline in the LD+ (4.3 [1.9-11.8]) than the LD- (1.0 [0.5-1.5]; P <.001) HIV+ patients. LFAT remained stable during follow-up in all groups. At follow-up, liver stiffness measured with TE was similar among all HIV, LD+, LD-, and control patients and between the LD+ and LD- patients measured with MRE. Advanced fibrosis by MRE was observed in 3 of LD+ and none of LD- patients. Conclusions: During 16 years of follow-up, progression of NAFLD is rare compared to development of T2DM in HIV+ patients.Peer reviewe

Hepatic ceramides dissociate steatosis and insulin resistance in patients with non-alcoholic fatty liver disease

Background & Aims: Recent data in mice have identified de novo ceramide synthesis as the key mediator of hepatic insulin resistance (IR) that in humans characterizes increases in liver fat due to IR ('Metabolic NAFLD' but not that due to the I148M gene variant in PNPLA3 ('PNPLA3 NAFLD'). We determined which bioactive lipids co-segregate with IR in the human liver. Methods: Liver lipidome was profiled in liver biopsies from 125 subjects that were divided into equally sized groups based on median HOMA-IR ('High and Low HOMA-IR', n = 62 and n = 63) or PNPLA3 genotype (PNPIA3(148MM/MI), n = 61 vs. PNPLA3(148II), n = 64). The subjects were also divided into 4 groups who had either IR, the I148M gene variant, both of the risk factors or neither. Results: Steatosis and NASH prevalence were similarly increased in 'High HOMA-IR' and PNPLA3(148MM/MI) groups compared to their respective control groups. The 'High HOMA-IR' but not the PNPLA3(148MM/MI) group had features of IR. The liver in 'High HOMA-IR' vs. low HOMA-IR' was markedly enriched in saturated and monounsaturated triacylglycerols and free fatty acids, dihydroceramides (markers of de novo ceramide synthesis) and ceramides. Markers of other ceramide synthetic pathways were unchanged. In PNPLA3(148MM/MI) vs. PNPLA3(148II), the increase in liver fat was due to polyunsaturated triacylglycerols while other lipids were unchanged. Similar changes were observed when data were analyzed using the 4 subgroups. Conclusions: Similar increases in liver fat and NASH are associated with a metabolically harmful saturated, ceramide-enriched liver lipidome in 'Metabolic NAFLD' but not in 'PNPLA3 NAFLD'. This difference may explain why metabolic but not PNPLA3 NAFLD increases the risk of type 2 diabetes and cardiovascular disease. (C) 2016 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.Peer reviewe

The MBOAT7 variant rs641738 alters hepatic phosphatidylinositols and increases severity of non-alcoholic fatty liver disease in humans

Non peer reviewe

Predictors of Liver Fat and Stiffness in Non-Alcoholic Fatty Liver Disease (NAFLD) - an 11-Year Prospective Study

Liver fat can be non-invasively measured by proton magnetic resonance spectroscopy (H-1-MRS) and fibrosis estimated as stiffness using transient elastography (FibroScan). There are no longitudinal data on changes in liver fat in Europids or on predictors of liver stiffness using these methods. We determined liver fat (1H-MRS) and clinical characteristics including features of insulin resistance at baseline and after a median follow-up period of 11.3 (range 7.3-13.4) years in 97 Finnish subjects. Liver stiffness was measured at 11.3 years. Liver fat content decreased by 5% (p <0.05) over time. Values at baseline and 11.3 years were closely interrelated (r = 0.81, p <0.001). Baseline liver fat (OR 1.32; 95% CI: 1.15-1.50) and change in BMI (OR 1.67; 95% CI: 1.24-2.25) were independent predictors of liver fat at 11.3 years (AUROC 0.90; 95% CI: 0.83-0.96). Baseline liver fat (AUROC 0.84; 95% CI: 0.76-0.92) predicted liver fat at 11.3 years more accurately than routinely available parameters (AUROC 0.76; 95% CI: 0.65-0.86, p = 0.02). At 11.3 years, 29% of the subjects had increased liver stiffness. Baseline liver fat (OR 2.17; 95% CI: 1.05-4.46) was an independent predictor of increased liver stiffness. These data show that liver fat is more important than the associated metabolic abnormalities as the predictor of future liver fat and fibrosis.Peer reviewe

MARC1 variant rs2642438 increases hepatic phosphatidylcholines and decreases severity of non-alcoholic fatty liver disease in humans

Non peer reviewe