Impact of fibre supplementation on microbiome and resilience in healthy participants: A randomized, placebo-controlled clinical trial.

BACKGROUND AND AIMS: The gut microbiome exerts important roles in health, e.g., functions in metabolism and immunology. These functions are often exerted via short-chain fatty acid (SCFA) production by gut bacteria. Studies demonstrating causal relationships between interventions targeting the microbiome and clinical outcomes are limited. This study aimed to show a causal relationship between microbiome modulation through fibre intervention and health. METHODS AND RESULTS: This randomized, double-blind, cross-over study included 65 healthy subjects, aged 45-70 years, with increased metabolic risk (i.e., body mass index [BMI] 25-30 kg/m2, low to moderate daily dietary fibre intake, <30g/day). Subjects took daily a fibre mixture of Acacia gum and carrot powder or placebo for 12 weeks, with an 8-week wash-out period. Faecal samples for measurement of SCFAs and microbiome analysis were collected every 4 weeks. Before and after each intervention period subjects underwent the mixed-meal PhenFlex challenge Test (PFT). Health effects were expressed as resilience to the stressors of the PFT and as fasting metabolic and inflammatory state. The fibre mixture exerted microbiome modulation, with an increase in ß-diversity (p < 0.001). α-diversity was lower during fibre mixture intake compared to placebo after 4, 8 and 12 weeks (p = 0.002; p = 0.012; p = 0.031). There was no effect observed on faecal SCFA concentrations, nor on any of the primary clinical outcomes (Inflammatory resilience: p = 0.605, Metabolic resilience: p = 0.485). CONCLUSION: Although the intervention exerted effects on gut microbiome composition, no effects on SCFA production, on resilience or fasting metabolic and inflammatory state were observed in this cohort. REGISTRATION NUMBER CLINICALTRIALS.GOV: NCT04829396.

Translatability of mouse muscle-aging for humans: the role of sex.

Muscle-aging drives sarcopenia and is a major public health issue. Mice are frequently used as a model for human muscle-aging, however, research investigating their translational value is limited. In addition, mechanisms underlying muscle-aging may have sex-specific features in humans, but it is not yet assessed whether these are recapitulated in mice. Here, we studied the effects of aging on a functional, histological and transcriptional level at multiple timepoints in male and female mice (4, 17, 21 and 25 months), with particular emphasis on sex-differences. The effects of natural aging on the transcriptome of quadriceps muscle were compared to humans on pathway level. Significant loss of muscle mass occurred late, at 25 months, in both male (-17%, quadriceps) and female mice (-10%, quadriceps) compared to young control mice. Concomitantly, we found in female, but not male mice, a slower movement speed in the aged groups compared to the young mice (P < 0.001). Consistently, weighted gene co-expression network analysis revealed a stronger association between the aging-related reduction of movement and aging-related changes in muscle transcriptome of female compared to male mice (P < 0.001). In male, but not female mice, major distinctive aging-related changes occurred in the last age group (25 months), which highlights the necessity for careful selection of age using mice as a muscle-aging model. Furthermore, contrasting to humans, more aging-related changes were found in the muscle transcriptome of male mice compared to female mice (4090 vs. 2285 differentially expressed genes at 25 months, respectively). Subsequently, male mice recapitulated more muscle-aging related pathways characteristic for both male and female humans. In conclusion, our data show that sex has a critical effect on the mouse muscle-aging trajectory, although these do not necessarily reflect sex differences observed in the human muscle-aging trajectory.

Corrigendum: Ldlr-/-.Leiden mice develop neurodegeneration, age-dependent astrogliosis and obesity-induced changes in microglia immunophenotype which are partly reversed by complement component 5 neutralizing antibody.

[This corrects the article DOI: 10.3389/fncel.2023.1205261.].

Evidence for sex-specific intramuscular changes associated to physical weakness in adults older than 75 years.

BACKGROUND: Physical weakness is a key component of frailty, and is highly prevalent in older adults. While females have a higher prevalence and earlier onset, sex differences in the development of frailty-related physical weakness are hardly studied. Therefore, we investigated the intramuscular changes that differentiate between fit and weak older adults for each sex separately. METHODS: Male (n = 28) and female (n = 26) older adults (75 + years) were grouped on the basis of their ranks according to three frailty-related physical performance criteria. Muscle biopsies taken from vastus lateralis muscle were used for transcriptome and histological examination. Pairwise comparisons were made between the fittest and weakest groups for each sex separately, and potential sex-specific effects were assessed. RESULTS: Weak females were characterized by a higher expression of inflammatory pathways and infiltration of NOX2-expressing immune cells, concomitant with a higher VCAM1 expression. Weak males were characterized by a smaller diameter of type 2 (fast) myofibers and lower expression of PRKN. In addition, weakness-associated transcriptome changes in the muscle were distinct from aging, suggesting that the pathophysiology of frailty-associated physical weakness does not necessarily depend on aging. CONCLUSIONS: We conclude that physical weakness-associated changes in muscle are sex-specific and recommend that sex differences are taken into account in research on frailty, as these differences may have a large impact on the development of (pharmaceutical) interventions against frailty. TRIAL REGISTRATION NUMBER: The FITAAL study was registered in the Dutch Trial Register, with registration code NTR6124 on 14-11-2016 ( https://trialsearch.who.int/Trial2.aspx?TrialID=NTR6124 ). HIGHLIGHTS: • In female, but not male older adults, physical weakness was associated with a higher expression of intramuscular markers for inflammation. • In male, but not female older adults, physical weakness was associated with a smaller diameter of type 2 (fast) myofibers and lower PRKN expression. • Fit older adults (of both sexes) maintained expression levels comparable to young participants of weakness related genes, differing from frail participants.

Ldlr-/-.Leiden mice develop neurodegeneration, age-dependent astrogliosis and obesity-induced changes in microglia immunophenotype which are partly reversed by complement component 5 neutralizing antibody.

Introduction: Obesity has been linked to vascular dysfunction, cognitive impairment and neurodegenerative diseases. However, experimental models that recapitulate brain pathology in relation to obesity and vascular dysfunction are still lacking. Methods: In this study we performed the histological and histochemical characterization of brains from Ldlr-/-.Leiden mice, an established model for obesity and associated vascular disease. First, HFD-fed 18 week-old and 50 week-old Ldlr-/-.Leiden male mice were compared with age-matched C57BL/6J mice. We then assessed the effect of high-fat diet (HFD)-induced obesity on brain pathology in Ldlr-/-.Leiden mice and tested whether a treatment with an anti-complement component 5 antibody, a terminal complement pathway inhibitor recently shown to reduce vascular disease, can attenuate neurodegeneration and neuroinflammation. Histological analyses were complemented with Next Generation Sequencing (NGS) analyses of the hippocampus to unravel molecular pathways underlying brain histopathology. Results: We show that chow-fed Ldlr-/-.Leiden mice have more severe neurodegeneration and show an age-dependent astrogliosis that is not observed in age-matched C57BL/6J controls. This was substantiated by pathway enrichment analysis using the NGS data which showed that oxidative phosphorylation, EIF2 signaling and mitochondrial dysfunction pathways, all associated with neurodegeneration, were significantly altered in the hippocampus of Ldlr-/-.Leiden mice compared with C57BL/6J controls. Obesity-inducing HFD-feeding did not aggravate neurodegeneration and astrogliosis in Ldlr-/-.Leiden mice. However, brains from HFD-fed Ldlr-/-.Leiden mice showed reduced IBA-1 immunoreactivity and increased CD68 immunoreactivity compared with chow-fed Ldlr-/-.Leiden mice, indicating alteration of microglial immunophenotype by HFD feeding. The systemic administration of an anti-C5 treatment partially restored the HFD effect on microglial immunophenotype. In addition, NGS data of hippocampi from Ldlr-/-.Leiden mice showed that HFD feeding affected multiple molecular pathways relative to chow-fed controls: HFD notably inactivated synaptogenesis and activated neuroinflammation pathways. The anti-C5 treatment restored the HFD-induced effect on molecular pathways to a large extent. Conclusion: This study shows that the Ldlr-/-.Leiden mouse model is suitable to study brain histopathology and associated biological processes in a context of obesity and provides evidence of the potential therapeutic value of anti-complement therapy against obesity-induced neuroinflammation.

Caloric Restriction Combined with Immobilization as Translational Model for Sarcopenia Expressing Key-Pathways of Human Pathology.

The prevalence of sarcopenia is increasing while it is often challenging, expensive and time-consuming to test the effectiveness of interventions against sarcopenia. Translational mouse models that adequately mimic underlying physiological pathways could accelerate research but are scarce. Here, we investigated the translational value of three potential mouse models for sarcopenia, namely partial immobilized (to mimic sedentary lifestyle), caloric restricted (CR; to mimic malnutrition) and a combination (immobilized & CR) model. C57BL/6J mice were calorically restricted (-40%) and/or one hindleg was immobilized for two weeks to induce loss of muscle mass and function. Muscle parameters were compared to those of young control (4 months) and old reference mice (21 months). Transcriptome analysis of quadriceps muscle was performed to identify underlying pathways and were compared with those being expressed in aged human vastus lateralis muscle-biopsies using a meta-analysis of five different human studies. Caloric restriction induced overall loss of lean body mass (-15%, p<0.001), whereas immobilization decreased muscle strength (-28%, p<0.001) and muscle mass of hindleg muscles specifically (on average -25%, p<0.001). The proportion of slow myofibers increased with aging in mice (+5%, p<0.05), and this was not recapitulated by the CR and/or immobilization models. The diameter of fast myofibers decreased with aging (-7%, p<0.05), and this was mimicked by all models. Transcriptome analysis revealed that the combination of CR and immobilization recapitulated more pathways characteristic for human muscle-aging (73%) than naturally aged (21 months old) mice (45%). In conclusion, the combination model exhibits loss of both muscle mass (due to CR) and function (due to immobilization) and has a remarkable similarity with pathways underlying human sarcopenia. These findings underline that external factors such as sedentary behavior and malnutrition are key elements of a translational mouse model and favor the combination model as a rapid model for testing the treatments against sarcopenia.

Semaglutide Has Beneficial Effects on Non-Alcoholic Steatohepatitis in Ldlr-/-.Leiden Mice.

Semaglutide, a glucagon-like peptide-1 receptor agonist, is an antidiabetic medication that has recently been approved for the treatment of obesity as well. Semaglutide is postulated to be a promising candidate for the treatment of non-alcoholic steatohepatitis (NASH). Here, Ldlr-/-.Leiden mice received a fast-food diet (FFD) for 25 weeks, followed by another 12 weeks on FFD with daily subcutaneous injections of semaglutide or vehicle (control). Plasma parameters were evaluated, livers and hearts were examined, and hepatic transcriptome analysis was performed. In the liver, semaglutide significantly reduced macrovesicular steatosis (-74%, p < 0.001) and inflammation (-73%, p < 0.001) and completely abolished microvesicular steatosis (-100%, p < 0.001). Histological and biochemical assessment of hepatic fibrosis showed no significant effects of semaglutide. However, digital pathology revealed significant improvements in the degree of collagen fiber reticulation (-12%, p < 0.001). Semaglutide did not affect atherosclerosis relative to controls. Additionally, we compared the transcriptome profile of FFD-fed Ldlr-/-.Leiden mice with a human gene set that differentiates human NASH patients with severe fibrosis from those with mild fibrosis. In FFD-fed Ldlr-/-.Leiden control mice, this gene set was upregulated as well, while semaglutide predominantly reversed this gene expression. Using a translational model with advanced NASH, we demonstrated that semaglutide is a promising candidate with particular potential for the treatment of hepatic steatosis and inflammation, while for the reversal of advanced fibrosis, combinations with other NASH agents may be necessary.

Atorvastatin Attenuates Diet-Induced Non-Alcoholic Steatohepatitis in APOE*3-Leiden Mice by Reducing Hepatic Inflammation.

Patients with metabolic syndrome are often prescribed statins to prevent the development of cardiovascular disease. Conversely, data on their effects on non-alcoholic steatohepatitis (NASH) are lacking. We evaluated these effects by feeding APOE*3-Leiden mice a Western-type diet (WTD) with or without atorvastatin to induce NASH and hepatic fibrosis. Besides the well-known plasma cholesterol lowering (-30%) and anti-atherogenic effects (severe lesion size -48%), atorvastatin significantly reduced hepatic steatosis (-22%), the number of aggregated inflammatory cells in the liver (-80%) and hepatic fibrosis (-92%) compared to WTD-fed mice. Furthermore, atorvastatin-treated mice showed less immunohistochemically stained areas of inflammation markers. Atorvastatin prevented accumulation of free cholesterol in the form of cholesterol crystals (-78%). Cholesterol crystals are potent inducers of the NLRP3 inflammasome pathway and atorvastatin prevented its activation, which resulted in reduced expression of the pro-inflammatory cytokines interleukin (IL)-1ß (-61%) and IL-18 (-26%). Transcriptome analysis confirmed strong reducing effects of atorvastatin on inflammatory mediators, including NLRP3, NFκB and TLR4. The present study demonstrates that atorvastatin reduces hepatic steatosis, inflammation and fibrosis and prevents cholesterol crystal formation, thereby precluding NLRP3 inflammasome activation. This may render atorvastatin treatment as an attractive approach to reduce NAFLD and prevent progression into NASH in dyslipidemic patients.

Translational characterization of the temporal dynamics of metabolic dysfunctions in liver, adipose tissue and the gut during diet-induced NASH development in Ldlr-/-.Leiden mice.

Background: NAFLD progression, from steatosis to inflammation and fibrosis, results from an interplay of intra- and extrahepatic mechanisms. Disease drivers likely include signals from white adipose tissue (WAT) and gut. However, the temporal dynamics of disease development remain poorly understood. Methods: High-fat-diet (HFD)-fed Ldlr-/-.Leiden mice were compared to chow-fed controls. At t = 0, 8, 16, 28 and 38w mice were euthanized, and liver, WAT depots and gut were analyzed biochemically, histologically and by lipidomics and transcriptomics together with circulating factors to investigate the sequence of pathogenic events and organ cross-talk during NAFLD development. Results: HFD-induced obesity was associated with an increase in visceral fat, plasma lipids and hyperinsulinemia at t = 8w, along with increased liver steatosis and circulating liver damage biomarkers. In parallel, upstream regulator analysis predicted that lipid catabolism regulators were deactivated and lipid synthesis regulators were activated. Subsequently, hepatocyte hypertrophy, oxidative stress and hepatic inflammation developed. Hepatic collagen accumulated from t = 16 w and became pronounced at t = 28-38 w. Epididymal WAT was maximally hypertrophic from t = 8 w, which coincided with inflammation development. Mesenteric and subcutaneous WAT hypertrophy developed slower and did not appear to reach a maximum, with minimal inflammation. In gut, HFD significantly increased permeability, induced a shift in microbiota composition from t = 8 w and changed circulating gut-derived metabolites. Conclusion: HFD-fed Ldlr-/-.Leiden mice develop obesity, dyslipidemia and insulin resistance, essentially as observed in obese NAFLD patients, underlining their translational value. We demonstrate that marked epididymal-WAT inflammation, and gut permeability and dysbiosis precede the development of NAFLD stressing the importance of a multiple-organ approach in the prevention and treatment of NAFLD.

Sex differences in skeletal muscle-aging trajectory: same processes, but with a different ranking.

Sex differences in muscle aging are poorly understood, but could be crucial for the optimization of sarcopenia-related interventions. To gain insight into potential sex differences in muscle aging, we recruited young (23 ± 2 years, 13 males and 13 females) and old (80 ± 3.5 years, 28 males and 26 females) participants. Males and females in both groups were highly matched, and vastus lateralis muscle parameters of old versus young participants were compared for each sex separately, focusing on gene expression. The overall gene expression profiles separated the sexes, but similar gene expression patterns separated old from young participants in males and females. Genes were indeed regulated in the same direction in both sexes during aging; however, the magnitude of differential expression was sex specific. In males, oxidative phosphorylation was the top-ranked differentially expressed process, and in females, this was cell growth mediated by AKT signaling. Findings from RNA-seq data were studied in greater detail using alternative approaches. In addition, we confirmed our data using publicly available data from three independent human studies. In conclusion, top-ranked pathways differ between males and females, but were present and altered in the same direction in both sexes. We conclude that the same processes are associated with skeletal muscle aging in males and females, but the differential expression of those processes in old vs. young participants is sex specific.

Unraveling the Transcriptional Dynamics of NASH Pathogenesis Affecting Atherosclerosis.

The prevalence of non-alcoholic steatohepatitis (NASH) is rapidly increasing and associated with cardiovascular disease (CVD), the major cause of mortality in NASH patients. Although sharing common risk factors, the mechanisms by which NASH may directly contribute to the development to CVD remain poorly understood. The aim of this study is to gain insight into key molecular processes of NASH that drive atherosclerosis development. Thereto, a time-course study was performed in Ldlr-/-.Leiden mice fed a high-fat diet to induce NASH and atherosclerosis. The effects on NASH and atherosclerosis were assessed and transcriptome analysis was performed. Ldlr-/-.Leiden mice developed obesity, hyperlipidemia and insulin resistance, with steatosis and hepatic inflammation preceding atherosclerosis development. Transcriptome analysis revealed a time-dependent increase in pathways related to NASH and fibrosis followed by an increase in pro-atherogenic processes in the aorta. Gene regulatory network analysis identified specific liver regulators related to lipid metabolism (SC5D, LCAT and HMGCR), inflammation (IL1A) and fibrosis (PDGF, COL3A1), linked to a set of aorta target genes related to vascular inflammation (TNFA) and atherosclerosis signaling (CCL2 and FDFT1). The present study reveals pathogenic liver processes that precede atherosclerosis development and identifies hepatic key regulators driving the atherogenic pathways and regulators in the aorta.

The Human Milk Oligosaccharide 2'-Fucosyllactose Alleviates Liver Steatosis, ER Stress and Insulin Resistance by Reducing Hepatic Diacylglycerols and Improved Gut Permeability in Obese Ldlr-/-.Leiden Mice.

Non-alcoholic fatty liver disease (NAFLD) is a complex multifactorial disorder that is associated with gut dysbiosis, enhanced gut permeability, adiposity and insulin resistance. Prebiotics such as human milk oligosaccharide 2'-fucosyllactose are thought to primarily improve gut health and it is uncertain whether they would affect more distant organs. This study investigates whether 2'-fucosyllactose can alleviate NAFLD development in manifest obesity. Obese hyperinsulinemic Ldlr-/-.Leiden mice, after an 8 week run-in on a high-fat diet (HFD), were treated with 2'-fucosyllactose by oral gavage until week 28 and compared to HFD-vehicle controls. 2'-fucosyllactose did not affect food intake, body weight, total fat mass or plasma lipids. 2'-fucosyllactose altered the fecal microbiota composition which was paralleled by a suppression of HFD-induced gut permeability at t = 12 weeks. 2'-fucosyllactose significantly attenuated the development of NAFLD by reducing microvesicular steatosis. These hepatoprotective effects were supported by upstream regulator analyses showing that 2'-fucosyllactose activated ACOX1 (involved in lipid catabolism), while deactivating SREBF1 (involved in lipogenesis). Furthermore, 2'-fucosyllactose suppressed ATF4, ATF6, ERN1, and NUPR1 all of which participate in endoplasmic reticulum stress. 2'-fucosyllactose reduced fasting insulin concentrations and HOMA-IR, which was corroborated by decreased intrahepatic diacylglycerols. In conclusion, long-term supplementation with 2'-fucosyllactose can counteract the detrimental effects of HFD on gut dysbiosis and gut permeability and attenuates the development of liver steatosis. The observed reduction in intrahepatic diacylglycerols provides a mechanistic rationale for the improvement of hyperinsulinemia and supports the use of 2'-fucosyllactose to correct dysmetabolism and insulin resistance.

Intervention with isoleucine or valine corrects hyperinsulinemia and reduces intrahepatic diacylglycerols, liver steatosis, and inflammation in Ldlr-/-.Leiden mice with manifest obesity-associated NASH.

Non-alcoholic steatohepatitis (NASH) is associated with a disturbed metabolism in liver, insulin resistance, and excessive accumulation of ectopic fat. Branched-chain amino acids (BCAAs) may beneficially modulate hepatic lipids, however, it remains unclear whether individual BCAAs can attenuate already established NASH and associated oxidative-inflammatory stress. After a 26 weeks run-in on fast food diet (FFD), obese Ldlr-/-.Leiden mice were treated for another 12 weeks with either valine or isoleucine (3% of FFD) and then compared to FFD controls. Valine and isoleucine did not affect obesity, dyslipidemia, gut permeability, or fecal fatty acid excretion, but significantly reduced hyperinsulinemia. Valine and isoleucine reduced ALT, CK18-M30, and liver steatosis with a particularly pronounced suppression of the microvesicular component (-61% by valine and -71% by isoleucine). Both BCAAs decreased intrahepatic diacylglycerols and 4-hydroxynonenal immunoreactivity, a marker for oxidative stress-induced lipid peroxidation. Functional genomics analysis demonstrated that valine and isoleucine affected BCAA metabolism genes, deactivated master regulators of anabolic pathways related to steatosis (e.g., SREBPF1), and activated master regulators of mitochondrial biogenesis (e.g., PPARGC1A) and lipid catabolism (e.g., ACOX1, AMPK). This correction of critical metabolic pathways on gene expression level was accompanied by a significant decrease in histological liver inflammation, and suppression of FFD-stimulated cytokine and chemokine proteins KC/CXCL1, MCP-1/CCL2, and MIP-2/CXCL2 and their pathways. In conclusion, dietary intervention with either valine or isoleucine corrected liver diacylglycerols, gene expression of multiple metabolic processes, and reduced NASH histology with profound hepatoprotective effects on oxidative stress and inflammatory proteins.

An altered microbiota pattern precedes Type 2 diabetes mellitus development: From the CORDIOPREV study.

Introduction: A distinctive gut microbiome have been linked to type 2 diabetes mellitus (T2DM). Objectives: We aimed to evaluate whether gut microbiota composition, in addition to clinical biomarkers, could improve the prediction of new incident cases of diabetes in patients with coronary heart disease. Methods: All the patients from the CORDIOPREV (Clinical Trials.gov.Identifier: NCT00924937) study without T2DM at baseline were included (n = 462). Overall, 107 patients developed it after a median of 60 months. The gut microbiota composition was determined by 16S rRNA gene sequencing and predictive models were created using hold-out method. Results: A gut microbiota profile associated with T2DM development was determined through a microbiome-based predictive model. The addition of microbiome data to clinical parameters (variables included in FINDRISC risk score and the diabetes risk score of the American Diabetes Association, HDL, triglycerides and HbA1c) improved the prediction increasing the area under the curve from 0.632 to 0.946. Furthermore, a microbiome-based risk score including the ten most discriminant genera, was associated with the probability of develop T2DM. Conclusion: These results suggest that a microbiota profile is associated to the T2DM development. An integrate predictive model of microbiome and clinical data that can improve the prediction of T2DM is also proposed, if is validated in independent populations to prevent this disease.

Mechanism-Based Biomarker Prediction for Low-Grade Inflammation in Liver and Adipose Tissue.

Metabolic disorders, such as obesity and type 2 diabetes have a large impact on global health, especially in industrialized countries. Tissue-specific chronic low-grade inflammation is a key contributor to complications in metabolic disorders. To support therapeutic approaches to these complications, it is crucial to gain a deeper understanding of the inflammatory dynamics and to monitor them on the individual level. To this end, blood-based biomarkers reflecting the tissue-specific inflammatory dynamics would be of great value. Here, we describe an in silico approach to select candidate biomarkers for tissue-specific inflammation by using a priori mechanistic knowledge from pathways and tissue-derived molecules. The workflow resulted in a list of candidate markers, in part consisting of literature confirmed biomarkers as well as a set of novel, more innovative biomarkers that reflect inflammation in the liver and adipose tissue. The first step of biomarker verification was on murine tissue gene-level by inducing hepatic inflammation and adipose tissue inflammation through a high-fat diet. Our data showed that in silico predicted hepatic markers had a strong correlation to hepatic inflammation in the absence of a relation to adipose tissue inflammation, while others had a strong correlation to adipose tissue inflammation in the absence of a relation to liver inflammation. Secondly, we evaluated the human translational value by performing a curation step in the literature using studies that describe the regulation of the markers in human, which identified 9 hepatic (such as Serum Amyloid A, Haptoglobin, and Interleukin 18 Binding Protein) and 2 adipose (Resistin and MMP-9) inflammatory biomarkers at the highest level of confirmation. Here, we identified and pre-clinically verified a set of in silico predicted biomarkers for liver and adipose tissue inflammation which can be of great value to study future development of therapeutic/lifestyle interventions to combat metabolic inflammatory complications.

Butyrate Protects against Diet-Induced NASH and Liver Fibrosis and Suppresses Specific Non-Canonical TGF-ß Signaling Pathways in Human Hepatic Stellate Cells.

In obesity-associated non-alcoholic steatohepatitis (NASH), persistent hepatocellular damage and inflammation are key drivers of fibrosis, which is the main determinant of NASH-associated mortality. The short-chain fatty acid butyrate can exert metabolic improvements and anti-inflammatory activities in NASH. However, its effects on NASH-associated liver fibrosis remain unclear. Putative antifibrotic effects of butyrate were studied in Ldlr-/-.Leiden mice fed an obesogenic diet (HFD) containing 2.5% (w/w) butyrate for 38 weeks and compared with a HFD-control group. Antifibrotic mechanisms of butyrate were further investigated in TGF-ß-stimulated primary human hepatic stellate cells (HSC). HFD-fed mice developed obesity, insulin resistance, increased plasma leptin levels, adipose tissue inflammation, gut permeability, dysbiosis, and NASH-associated fibrosis. Butyrate corrected hyperinsulinemia, lowered plasma leptin levels, and attenuated adipose tissue inflammation, without affecting gut permeability or microbiota composition. Butyrate lowered plasma ALT and CK-18M30 levels and attenuated hepatic steatosis and inflammation. Butyrate inhibited fibrosis development as demonstrated by decreased hepatic collagen content and Sirius-red-positive area. In TGF-ß-stimulated HSC, butyrate dose-dependently reduced collagen deposition and decreased procollagen1α1 and PAI1 protein expression. Transcriptomic analysis and subsequent pathway and upstream regulator analysis revealed deactivation of specific non-canonical TGF-ß signaling pathways Rho-like GTPases and PI3K/AKT and other important pro-fibrotic regulators (e.g., YAP/TAZ, MYC) by butyrate, providing a potential rationale for its antifibrotic effects. In conclusion, butyrate protects against obesity development, insulin resistance-associated NASH, and liver fibrosis. These antifibrotic effects are at least partly attributable to a direct effect of butyrate on collagen production in hepatic stellate cells, involving inhibition of non-canonical TGF-ß signaling pathways.

Diet and exercise reduce pre-existing NASH and fibrosis and have additional beneficial effects on the vasculature, adipose tissue and skeletal muscle via organ-crosstalk.

BACKGROUND: Non-alcoholic steatohepatitis (NASH) has become one of the most common liver diseases and is still without approved pharmacotherapy. Lifestyle interventions using exercise and diet change remain the current treatment of choice and even a small weight loss (5-7%) can already have a beneficial effect on NASH. However, the underlying molecular mechanisms of exercise and diet interventions remain largely elusive, and it is unclear whether they exert their health effects via similar or different pathways. METHODS: Ldlr-/-.Leiden mice received a high fat diet (HFD) for 30 weeks to establish a severe state of NASH/fibrosis with simultaneous atherosclerosis development. Groups of mice were then either left untreated (control group) or were treated for 20 weeks with exercise (running wheel), diet change (switch to a low fat chow diet) or the combination thereof. The liver and distant organs including heart, white adipose tissue (WAT) and muscle were histologically examined. Comprehensive transcriptome analysis of liver, WAT and muscle revealed the organ-specific effects of exercise and diet and defined the underlying pathways. RESULTS: Exercise and dietary change significantly reduced body weight, fat mass, adipocyte size and improved myosteatosis and muscle function with additive effects of combination treatment. WAT inflammation was significantly improved by diet change, tended to be reduced with exercise, and combination therapy had no additive effect. Hepatic steatosis and inflammation were almost fully reversed by exercise and diet change, while hepatic fibrosis tended to be improved with exercise and was significantly improved with diet change. Additive effects for the combination therapy were shown for liver steatosis and associated liver lipids, and atherosclerosis, but not for hepatic inflammation and fibrosis. Pathway analysis revealed complementary effects on metabolic pathways and lipid handling processes, thereby substantiating the added value of combined lifestyle treatment. CONCLUSIONS: Exercise, diet change and the combination thereof can reverse established NASH/fibrosis in obese Ldlr-/-.Leiden mice. In addition, the lifestyle interventions had beneficial effects on atherosclerosis, WAT inflammation and muscle function. For steatosis and other parameters related to adiposity or lipid metabolism, exercise and dietary change affected more distinct pathways that acted complementary when the interventions were combined resulting in an additive effect for the combination therapy on important endpoints including NASH and atherosclerosis. For inflammation, exercise and diet change shared several underlying pathways resulting in a net similar effect when the interventions were combined.

Krill Oil Treatment Increases Distinct PUFAs and Oxylipins in Adipose Tissue and Liver and Attenuates Obesity-Associated Inflammation via Direct and Indirect Mechanisms.

The development of obesity is characterized by the metabolic overload of tissues and subsequent organ inflammation. The health effects of krill oil (KrO) on obesity-associated inflammation remain largely elusive, because long-term treatments with KrO have not been performed to date. Therefore, we examined the putative health effects of 28 weeks of 3% (w/w) KrO supplementation to an obesogenic diet (HFD) with fat derived mostly from lard. The HFD with KrO was compared to an HFD control group to evaluate the effects on fatty acid composition and associated inflammation in epididymal white adipose tissue (eWAT) and the liver during obesity development. KrO treatment increased the concentrations of EPA and DHA and associated oxylipins, including 18-HEPE, RvE2 and 14-HDHA in eWAT and the liver. Simultaneously, KrO decreased arachidonic acid concentrations and arachidonic-acid-derived oxylipins (e.g., HETEs, PGD2, PGE2, PGF2α, TXB2). In eWAT, KrO activated regulators of adipogenesis (e.g., PPARγ, CEBPα, KLF15, STAT5A), induced a shift towards smaller adipocytes and increased the total adipocyte numbers indicative for hyperplasia. KrO reduced crown-like structures in eWAT, and suppressed HFD-stimulated inflammatory pathways including TNFα and CCL2/MCP-1 signaling. The observed eWAT changes were accompanied by reduced plasma leptin and increased plasma adiponectin levels over time, and improved insulin resistance (HOMA-IR). In the liver, KrO suppressed inflammatory signaling pathways, including those controlled by IL-1ß and M-CSF, without affecting liver histology. Furthermore, KrO deactivated hepatic REL-A/p65-NF-κB signaling, consistent with increased PPARα protein expression and a trend towards an increase in IkBα. In conclusion, long-term KrO treatment increased several anti-inflammatory PUFAs and oxylipins in WAT and the liver. These changes were accompanied by beneficial effects on general metabolism and inflammatory tone at the tissue level. The stimulation of adipogenesis by KrO allows for safe fat storage and may, together with more direct PPAR-mediated anti-inflammatory mechanisms, attenuate inflammation.

A Novel Personalized Systems Nutrition Program Improves Dietary Patterns, Lifestyle Behaviors and Health-Related Outcomes: Results from the Habit Study.

Personalized nutrition may be more effective in changing lifestyle behaviors compared to population-based guidelines. This single-arm exploratory study evaluated the impact of a 10-week personalized systems nutrition (PSN) program on lifestyle behavior and health outcomes. Healthy men and women (n = 82) completed the trial. Individuals were grouped into seven diet types, for which phenotypic, genotypic and behavioral data were used to generate personalized recommendations. Behavior change guidance was also provided. The intervention reduced the intake of calories (-256.2 kcal; p < 0.0001), carbohydrates (-22.1 g; p < 0.0039), sugar (-13.0 g; p < 0.0001), total fat (-17.3 g; p < 0.0001), saturated fat (-5.9 g; p = 0.0003) and PUFA (-2.5 g; p = 0.0065). Additionally, BMI (-0.6 kg/m2; p < 0.0001), body fat (-1.2%; p = 0.0192) and hip circumference (-5.8 cm; p < 0.0001) were decreased after the intervention. In the subgroup with the lowest phenotypic flexibility, a measure of the body's ability to adapt to environmental stressors, LDL (-0.44 mmol/L; p = 0.002) and total cholesterol (-0.49 mmol/L; p < 0.0001) were reduced after the intervention. This study shows that a PSN program in a workforce improves lifestyle habits and reduces body weight, BMI and other health-related outcomes. Health improvement was most pronounced in the compromised phenotypic flexibility subgroup, which indicates that a PSN program may be effective in targeting behavior change in health-compromised target groups.

Cholesterol Accumulation as a Driver of Hepatic Inflammation Under Translational Dietary Conditions Can Be Attenuated by a Multicomponent Medicine.

Background: Non-alcoholic fatty liver disease (NAFLD) is a complex multifactorial disorder that is characterised by dysfunctional lipid metabolism and cholesterol homeostasis, and a related chronic inflammatory response. NAFLD has become the most common cause of chronic liver disease in many countries, and its prevalence continues to rise in parallel with increasing rates of obesity. Here, we evaluated the putative NAFLD-attenuating effects of a multicomponent medicine consisting of 24 natural ingredients: Hepar compositum (HC-24). Methods: Ldlr-/-.Leiden mice were fed a high-fat diet (HFD) with a macronutrient composition and cholesterol content comparable to human diets for 24 weeks to induce obesity-associated metabolic dysfunction, including hepatic steatosis and inflammation. HC-24 or vehicle control was administered intraperitoneally 3 times/week (1.5 ml/kg) for the last 18 weeks of the study. Histological analyses of liver and adipose tissue were combined with extensive hepatic transcriptomics analysis. Transcriptomics results were further substantiated with ELISA, immunohistochemical and liver lipid analyses. Results: HFD feeding induced obesity and metabolic dysfunction including adipose tissue inflammation and increased gut permeability. In the liver, HFD-feeding resulted in a disturbance of cholesterol homeostasis and an associated inflammatory response. HC-24 did not affect body weight, metabolic risk factors, adipose tissue inflammation or gut permeability. While HC-24 did not alter total liver steatosis, there was a pronounced reduction in lobular inflammation in HC-24-treated animals, which was associated with modulation of genes and proteins involved in inflammation (e.g., neutrophil chemokine Cxcl1) and cholesterol homeostasis (i.e., predicted effect on 'cholesterol' as an upstream regulator, based on gene expression changes associated with cholesterol handling). These effects were confirmed by CXCL1 ELISA, immunohistochemical staining of neutrophils and biochemical analysis of hepatic free cholesterol content. Intrahepatic free cholesterol levels were found to correlate significantly with the number of inflammatory aggregates in the liver, thereby providing a potential rationale for the observed anti-inflammatory effects of HC-24. Conclusions: Free cholesterol accumulates in the liver of Ldlr-/-.Leiden mice under physiologically translational dietary conditions, and this is associated with the development of hepatic inflammation. The multicomponent medicine HC-24 reduces accumulation of free cholesterol and has molecular and cellular anti-inflammatory effects in the liver.