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

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

Effects of Weighted Hula-Hooping Compared to Walking on Abdominal Fat, Trunk Muscularity, and Metabolic Parameters in Overweight Subjects : A Randomized Controlled Study

Background: Weighted hula-hoops have gained popularity, but whether they indeed reshape the trunk or have beneficial metabolic effects in overweight subjects is unknown. Objectives: To determine effects of hula-hooping and walking matched for energy expenditure on android fat %, trunk muscle mass, and metabolic parameters in a randomized cross-over study. Design: We recruited 55 overweight nondiabetic subjects, who were randomized to hula-hooping (HULA) for 6 weeks using a 1.5-kg weighted hula-hoop followed by walking (WALK) for another 6 weeks or vice versa. The increments in energy expenditure were similar by HULA and WALK. Body composition (dual-energy X-ray absorptiometry) and metabolic parameters were measured at baseline and after HULA and WALK. The primary endpoint was the change in fat % in the android region. Results: A total of 53subjects (waist 92 +/- 1 cm, body mass index 28 +/- 1 kg/m(2)) completed the study. Body weight changed similarly (-0.6 +/- 0.2 vs. -0.5 +/- 0.2 kg, nonsignificant; HULA vs. WALK). During the intervention the subjects hula-hooped on average 12.8 +/- 0.5 min/day and walked 9,986 +/- 376 steps/day. The % fat in the android region decreased significantly by HULA but not by WALK (between-group change p <0.001). Trunk muscle mass increased more by HULA than by WALK (p <0.05). Waist circumference decreased more by HULA than by WALK (-3.1 +/- 0.3 cm vs. -0.7 +/- 0.4 cm, p <0.001; HULA vs. WALK). WALK but not HULA significantly lowered systolic blood pressure and increased HDL cholesterol while HULA significantly decreased LDL cholesterol. Conclusions: Hula-hooping with a weighted hula-hoop can be used to decrease abdominal fat % and increase trunk muscle mass in overweight subjects. Its LDL lowering effect resembles that described for resistance training. (c) 2019 The Author(s) Published by S. Karger AG, BaselPeer reviewe

MicroRNA-192*impairs adipocyte triglyceride storage

We investigated the expression of miR-192* (miR-192-3p) in the visceral adipose tissue (VAT) of obese subjects and its function in cultured human adipocytes. This miRNA is a 3' arm derived from the same pre-miRNA as miR-192 (miR-192-5p) implicated in type 2 diabetes, liver disease and cancers, and is predicted to target key genes in lipid metabolism. In morbidly obese subjects undergoing bariatric surgery preceded by a very low calorie diet, miR-192* in VAT correlated negatively (r = -0.387; p = 0.046) with serum triglyceride (TG) and positively with high-density lipoprotein (HDL) concentration (r = 0.396; p = 0.041). In a less obese patient cohort, the miRNA correlated negatively with the body mass index (r = -0.537; p = 0.026). To characterize the function of miR-192*, we overexpressed it in cultured adipocytes and analyzed the expression of adipogenic differentiation markers as well as cellular TG content. Reduced TG and expression of the adipocyte marker proteins aP2 (adipocyte protein 2) and perilipin 1 were observed. The function of miR-192* was further investigated by transcriptomic profiling of adipocytes expressing this miRNA, revealing impacts on key lipogenic genes. A number of the mRNA alterations were validated by qPCR. Western analysis confirmed a marked reduction of the lipogenic enzyme SCD (stearoyl coenzyme A desaturase-1), the fatty aldehyde dehydrogenase ALDH3A2 (aldehyde dehydrogenase 3 family member A2) and the high-density lipoprotein receptor SCARB1 (scavenger receptor B, type I). SCD and ALDH3A2 were demonstrated to be direct targets of miR-192*. To conclude, the present data identify miR-192* as a novel controller of adipocyte differentiation and lipid homeostasis. (C) 2016 Elsevier B.V. All rights reserved.Peer reviewe

Saturated fat is more metabolically harmful for the human liver than unsaturated fat or simple sugars

OBJECTIVE Nonalcoholic fatty liver disease (i.e., increased intrahepatic triglyceride [IHTG] content), predisposes to type 2 diabetes and cardiovascular disease. Adipose tissue lipolysis and hepatic de novo lipogenesis (DNL) are the main pathways contributing to IHTG. We hypothesized that dietary macronutrient composition influences the pathways, mediators, and magnitude of weight gain-induced changes in IHTG. RESEARCH DESIGN AND METHODS We overfed 38 overweight subjects (age 48 ± 2, BMI 31 ± 1 kg/m2, liver fat 4.7 ± 0.9%) 1,000 extra kcal/day of saturated (SAT) or unsaturated (UNSAT) fat or simple sugars (CARB) for 3 weeks. We measured IHTG (1H-MRS), pathways contributing to IHTG (lipolysis ([2H5]glycerol) and DNL (2H2O) basally and during euglycemic hyperinsulinemia, insulin resistance, endotoxemia, plasma ceramides, and adipose tissue gene expression at 0 and 3 weeks. RESULTS Overfeeding SAT increased IHTG more (+55%) than UNSAT (+15%, P < 0.05). CARB increased IHTG (+33%) by stimulating DNL (+98%). SAT significantly increased while UNSAT decreased lipolysis. SAT induced insulin resistance and endotoxemia and significantly increased multiple plasma ceramides. The diets had distinct effects on adipose tissue gene expression. CONCLUSIONS Macronutrient composition of excess energy influences pathways of IHTG: CARB increases DNL, while SAT increases and UNSAT decreases lipolysis. SAT induced greatest increase in IHTG, insulin resistance, and harmful ceramides. Decreased intakes of SAT could be beneficial in reducing IHTG and the associated risk of diabetes

Regulation of Angiopoietin-Like Proteins (ANGPTLs) 3 and 8 by Insulin

Objective: Circulating ANGPTL8 has recently been used as a marker of insulin action. We studied expression and insulin regulation of ANGPTL8 and ANGPTL3 in vivo and in vitro. Design and Methods: Expression of ANGPTL8 and ANGPTL3 was studied in 34 paired samples of human liver and adipose tissue. Effects of insulin on 1) plasma concentrations and adipose tissue expression of ANGPTL8 and ANGPTL3 (in vivo 6-h euglycemic hyperinsulinemia; n = 18), and 2) ANGPTL8 and ANGPTL3 gene and protein expression in immortalized human hepatocytes (IHH) and adipocytes were measured. Effect of ANGPTL3 on secretion of ANGPTL8 in cells stably over-expressing ANGPTL3, -8, or both was determined. Results: ANGPTL3 was only expressed in the liver, whereas ANGPTL8 was expressed in both tissues. In vivo hyperinsulinemia significantly decreased both plasma ANGPTL8 and ANGPTL3 at 3 and 6 hours. Insulin increased ANGPTL8 expression in human adipose tissue 14- and 18-fold at 3 and 6 hours and ANGPTL8 was the most insulin-responsive transcript on microarray. Insulin also increased ANPGTL8 in cultured adipocytes and IHH but the protein mainly remained intracellular. In vitro in IHH, insulin decreased ANGPTL3 gene expression and secretion of ANGPTL3 into growth medium. Overexpression of ANGPTL8 in CHO cells did not result in its release into culture medium while abundant secretion occurred in cells co-expressing ANGPTL3 and -8. Conclusions: Insulin decreases plasma ANGPTL3 by decreasing ANGPTL3 expression in the liver. Insulin markedly increases ANGPTL8 in adipose tissue and the liver but not in plasma. These data show that measurement of plasma ANGPTL3 but not -8 reflects insulin action in target tissues.Peer reviewe

Overfeeding saturated fat increases LDL (low-density lipoprotein) aggregation susceptibility while overfeeding unsaturated fat decreases proteoglycan-binding of lipoproteins

Publisher Copyright: © 2021 The Authors.OBJECTIVE: We recently showed that measurement of the susceptibility of LDL (low-density lipoprotein) to aggregation is an independent predictor of cardiovascular events. We now wished to compare effects of overfeeding different dietary macronutrients on LDL aggregation, proteoglycan-binding of plasma lipoproteins, and on the concentration of oxidized LDL in plasma, 3 in vitro parameters consistent with increased atherogenicity. APPROACH AND RESULTS: The participants (36 subjects; age, 48±10 years; body mass index, 30.9±6.2 kg/m2) were randomized to consume an extra 1000 kcal/day of either unsaturated fat, saturated fat, or simple sugars (CARB) for 3 weeks. We measured plasma proatherogenic properties (susceptibility of LDL to aggregation, proteoglycan-binding, oxidized LDL) and concentrations and composition of plasma lipoproteins using nuclear magnetic resonance spectroscopy, and in LDL using liquid chromatography mass spectrometry, before and after the overfeeding diets. LDL aggregation increased in the saturated fat but not the other groups. This change was associated with increased sphingolipid and saturated triacylglycerols in LDL and in plasma and reduction of clusterin on LDL particles. Proteoglycan binding of plasma lipoproteins decreased in the unsaturated fat group relative to the baseline diet. Lipoprotein properties remained unchanged in the CARB group. CONCLUSIONS: The type of fat during 3 weeks of overfeeding is an important determinant of the characteristics and functional properties of plasma lipoproteins in humans.Peer reviewe

Novel hepatic microRNAs upregulated in human nonalcoholic fatty liver disease

MicroRNAs (miRNAs) control gene expression by reducing mRNA stability and translation. We aimed to identify alterations in human liver miRNA expression/function in nonalcoholic fatty liver disease (NAFLD). Subjects with the highest (median liver fat 30%, n = 15) and lowest (0%, n = 15) liver fat content were selected from >100 obese patients for miRNA profiling of liver biopsies on microarrays carrying probes for 1438 human miRNAs (a cross-sectional study). Target mRNAs and pathways were predicted for the miRNAs most significantly upregulated in NAFLD, their cell-type-specific expression was investigated by quantitative PCR (qPCR), and the transcriptome of immortalized human hepatocytes (IHH) transfected with the miRNA with the highest number of predicted targets, miR-576-5p, was studied. The screen revealed 42 miRNAs up- and two downregulated in the NAFLD as compared to non-NAFLD liver. The miRNAs differing most significantly between the groups, miR-103a-2*, miR-106b, miR-576-5p, miRPlus-I137*, miR-892a, miR-1282, miR-3663-5p, and miR-3924, were all upregulated in NAFLD liver. Target pathways predicted for these miRNAs included ones involved in cancer, metabolic regulation, insulin signaling, and inflammation. Consistent transcriptome changes were observed in IHH transfected with miR-576-5p, and western analysis revealed a marked reduction of the RAC1 protein belonging to several miR-576-5p target pathways. To conclude, we identified 44 miRNAs differentially expressed in NAFLD versus non-NAFLD liver, 42 of these being novel in the context of NAFLD. The study demonstrates that by applying a novel study set-up and a broad-coverage array platform one can reveal a wealth of previously undiscovered miRNA dysregulation in metabolic disease.Peer reviewe

Continuous Grading of Early Fibrosis in NAFLD Using Label-Free Imaging : A Proof-of- Concept Study

Maksan rasvoittumisen ajatellaan joskus olevan vain metabolisen oireyhtymän hyvälaatuinen maksamanifestaatio, vaikka se johtaa, erityisesti maksan fibrotisoituessa, kasvaneeseen kardiovaskulaarikuolleisuuteen ja pahimmillaan maksasyöpään. Tästä kansanterveydellisesti merkittävästä roolista huolimatta rasvamaksa on alidiagnosoitu ja sen patogeneettiset mekanismit ovat huonosti ymmärrettyjä, molemmat osin hyvien diagnostisten työkalujen puutteesta johtuen. Päätimme selvittää uusien non-lineaaristen kuvantamismodaliteettien soveltuvuutta rasvamaksan varhaisessa diagnostiikassa. Selvitimme 32 lihavuusleikkauspotilaan maksakudoskoepalojen kudosarkkitehtuurin perinteisin kudosvärjäysmenetelmin ja vertasimme tuloksia samojen potilaiden non-lineaarisesti kuvannettuihin näytteisiin. Kuvansimme minimaalisesti käsitellyistä jääleikkeistä coherent anti-Stokes Raman scattering (CARS) ja second harmonic generation (SHG) signaalit, joita hyödyntäen arvioimme näytteiden rasva- ja kollageenipitoisuuden. Kehitimme myös kuva-analytiikka-algoritmin, joka mahdollistaa kollageenisignaalin automatisoidun ja objektiivisen arvioimisen. Näytteistä mittaamamme SHG- ja CARS-signaalit korreloivat hyvin patologien arvioihin näytteiden fibrotisoitumis- ja rasvoittumisasteista, minkä lisäksi automatisoitu algoritmimme erotteli luotettavasti jopa varhaisen fibroosin normaalista kudoksesta. Myös terveiksi luokiteltujen näytteiden maksakudoksen keskellä esiintyi hienoa fibrillaarista SHG-signaalia. Tunnistamamme SHG-signaali oli spesifiä kolokalisoituen hyvin kollageeni-I- ja III-vasta-ainevärjäyksien kanssa. Tuloksemme osoittavat, että multimodaalisten kuvantamismenetelmien avulla voidaan tunnistaa maksan fibrillaarista kollageenia perinteisiä kudostutkimusmenetelmiä herkemmin. Kehittämämme kuva-analyysi-algoritmi puolestaan arvioi varhaisen fibroosin määrän nopeasti, objektiivisesti ja jatkuvalla skaalalla. Yhdessä nämä löydökset luovat edellytykset tutkia esimerkiksi maksan vähäisen fibroosin kliinistä merkitystä ja paranemistaipumusta isommissa potilasaineistoissa