Introduction

The gut Lactobacillaceae have been shown to drive the onset of fatty liver disease¹, through a mechanism by which dietary carbohydrates are fermented into ethanol. Increased levels of Lactobacillaceae provoke high levels of portal blood ethanol and in turn promote the development of metabolic dysfunction-associated fatty liver disease (MAFLD) ^{1,2, 3}.Although the potential importance of ethanol production by the microflora is well recognized, theimplications on the clinical course of MAFLD remain obscure at best. A systematic studycomparing Lactobacillaceae levels and liver condition has not been performed. Furthermore,while lifestyle intervention is the mainstay in the clinical management of MAFLD, its effects on thelevels of Lactobacillaceae remain unknown, prompting investigations in this respect. Such studies,however, are complicated by the potential consumption of alcoholic beverages by the studysubjects involved¹, hampering the interpretation of results.

Aims & Methods

We examine the relationship of the Lactobacillaceae with circulating risk biomarkers for MAFLD in alcohol-free-use individuals (for religious reasons), who underwent time-restricted food intake (Ramadan). Microbiome determinations were performed by bacterial 16S rRNA sequencing.

Results

A total of 271 fecal samples were longitudinally collected from 93 individuals who subjectedthemselves to 30 days of intermittent fasting for religious reasons (a cohort of healthy volunteers[n = 35]; a cohort of morbidly obese individuals [n = 18]; and a cohort of patients with type 2diabetes [n = 40]). The mean age of the cohort was 44.3 years (SD = 8.10), consisting of 51.6% males(n = 48). Firstly, we observed in the participants that intermittent fasting is not effective in influencing the levels of Lactobacillaceae in all three cohorts (one-way ANOVA followed by multiple comparisons usingBonferroni corrections, P_adj > 0.2 for all intra-comparisons). Strikingly, however, when compared to healthy controls, a significant fraction of patients with type 2 diabetes shows high levels of alcohol-producing Lactobacillacea (p < 0.0001) suggesting that such bacteria contribute to the onset of disease. Furthermore, a significant correlation between GGT levels and the Lactobacillaceae component in the microbiome was observed (R = 0.18, P = 0.002). Interestingly, significant correlations of this bacterial with both glucose (R = 0.32, P < 0.0001) and HbA1c (R = 0.28, P < 0.0001) were also observed, in line with the notion that hepatic insulin resistance induced by the gut microbiome-derived ethanol is an important contributor to liver damage in the context of MAFLD^{3, 5}. In a multivariable model, glucose levels were shown to have the largest effect size on the variation of the Lacbotacillaceae community (R² = 1.60 %, p_adj < 0.001), with age contributing an additional 1.06% to the Lactobacillaceae microbiome variation (P_adj = 0.004). GGT tends to exert a moderate contribution to the variation of this community (P_adj = 0.099). However, intermittent fasting as a prominent external lifestyle factor is not identified as a significant contributor in this respect (P_adj = 0.254).

Conclusion

Overall, while the Lactobacillaceae clearly emerges as a driver of liver damage in alcohol-abstaining individuals, our data suggest that lifestyle intervention is not sufficient to counteractLactobacillaceae in MALFD calling for alternative approaches in the management of the patientsinvolved.

References

1. Meijnikman AS, et al. Nat Med 2022;28:2100-2106.
2. Fan Y, et al. Gut microbiota in human metabolic health and disease. Nat Rev Microbiol2021;19:55-71.
3. Zhu L, et al. Gut 2016;65:1232.
4. Su J, Wang Y, et al. Am J Clin Nutr 2021;113:1332-1342.
5. Engstler AJ, et al. Gut 2016;65:1564-71.6. Oksanen J. vegan: Community Ecology Package-R package version 1.17-8. http://CRAN.R-project. org/package= vegan 2011

Introduction

Metabolic dysfunction-associated steatotic liver disease (MASLD) and metabolic dysfunction with alcohol-related steatotic liver disease (MetALD) are two separate entities within the spectrum of steatotic liver disease (SLD).

Aims & Methods

This meta-analysis aims to compare the risks of hepatic and extrahepatic outcomes between MASLD and MetALD. We systematically searched for observational cohort studies published up to March 1, 2025, comparing clinical outcomes between individuals with MASLD and MetALD. The primary outcome was liver-related events, while secondary outcomes included hepatocellular carcinoma (HCC), liver-related mortality, cardiovascular events, extrahepatic cancers, and all-cause mortality. We used random-effect models to calculate pooled hazard ratios (HRs) with 95% confidence intervals (95% CI).

Results

Twenty-four studies involving 11,396,962 individuals were analyzed (9,622,716 with MASLD and 1,774,246 with MetALD). Individuals with MetALD had significantly higher risks of liver-related events (HR 1.62, 95% CI 1.16-2.25, p=0.02) and HCC (HR 1.33, 95%CI 1.00-1.77, p=0.04) than those with MASLD. The risk of extrahepatic cancers was also increased in MetALD (HR 1.03, 95% CI 1.01-1.06, p<0.001), while the rates of cardiovascular events (HR 0.96, 95% CI 0.85-1.09, p=0.48), extrahepatic cancer-related mortality (HR 1.44, 95% CI 0.97-2.15, p=0.07), and all-cause mortality (HR 1.08, 95% CI 0.97-1.19, p=0.14) did not differ between these two conditions.

Conclusion

MetALD confers a higher risk of liver-related outcomes, HCC, and extrahepatic cancers than MASLD, while all-cause mortality and cardiovascular risk are similar between the two conditions. These findings emphasize the need for distinct clinical strategies for these related yet different entities within the SLD spectrum and highlight the importance of conducting pharmacological clinical trials in this context.

Disclosure

Nothing to disclose

Introduction

Metabolic dysfunction-associated fatty liver disease (MAFLD), typically associated with obesity, is the leading cause of reversible insulin resistance, hepatic steatosis, and inflammation, which can be further progressed into unreversible fibrosis and finally liver failure when left without intervention. The necessity of timely intervention, especially changes in lifestyle, is well-recognized but is hampered by the lack of understanding of the molecular mechanisms involved.
A potential mediator of the beneficial effects of lifestyle change (less fat and carbohydrate intake, more physical exercise) is the gut microbiome¹. Lower amounts of intestinal bacteria producing short-chain fatty acids, especially members of the Lachnospiraceae and Ruminococcaceae family, have been closely associated with MAFLD and fibrosis progression^1-3, supporting the causality of the above associations.
Among the lifestyle changes suggested to counteract MAFLD, is intermittent fasting (IF)^4,5. IF is a popular dietary regimen and is typically defined by repeated temporal abstinence of food intake for 12 to 24 hours⁶. The microbiome partly mediates the potential effects of IF on liver physiology, as shown in previous studies in which we showed that one month of 16 hours daily fasting (OMIF) increases the Lachnospiraceae and Ruminococcaceae families in non-obese human volunteers⁷ as well as in healthy mice⁸. Importantly, we observed that OMIF provokes weight loss and reduces GGT levels even in healthy volunteers⁷. We, therefore, hypothesized that OMIF has beneficial effects and can potentially reverse MAFLD through modulation of the microbiome composition.

Aims & Methods

The aim of the current study was to investigate the effects of OMIF on MAFLD phenotypes and especially how changes in the microbiome influence the results. Experimental OMIF in high-fat diet-induced iC57BL/6J mice was investigated, focusing on the change of insulin resistance, liver function biomarkers (AST [aspartate aminotransferase], ALT [alanine transaminase], GGT [gamma-glutamyl tansferase] and triglycerides) and histopathology, which were correlated to the gut microbiome determined by 16S rRNA-sequencing. The protective potential of OMIF against MALFD was prospectively studied in obese volunteers. Fecal microbiota transplantation was applied to pseudo-germ-free mice. Microbial metabolite effects were tested in human MAFLD liver-derived organoids.

Results

Experimental OMIF decreased insulin resistance (p<0.001), AST, ALT, and triglycerides levels (all p<0.05), and especially improved liver pathology by 63.0% (p<0.001), as indicated by steatosis and inflammation. The results correlated with Lachnospiraceae and butyrate-producing genera, both increased by 46.9% (p=0.003) and 63.0 % (p=0.001), respectively. In obese human volunteers, OMIF caused weight loss by 3.5% (p=0.003) and improved all liver function biomarkers (p<0.001). This again correlated with Lachnospiraceae and butyrate-producing genera, both increased by 53.6% (p=0.0005) and 28.4% (p=0.001), respectively. Transplantation of stool from either OMIF-treated mice or humans led to improved insulin sensitivity, liver function, and liver pathology (all p<0.05) in recipient mice. When exposed to butyrate, the MAFLD-mimicking liver organoids showed less microvesicular fatty accumulation (p<0.001).

Conclusion

Manipulation of the gut microbiome by OMIF can contract MAFLD. The Lachnospiraceae and butyrate-producing genera-enriched communities should be considered as new therapeutic targets in MAFLD.

References

1. Asrani SK, Devarbhavi H, Eaton J, et al. Burden of liver diseases in the world. J Hepatol 2019;70:151-171.
2. Canfora EE, Meex RCR, Venema K, et al. Gut microbial metabolites in obesity, NAFLD and T2DM. Nat Rev Endocrinol 2019;15:261-273.
3. Flint HJ, Duncan SH, Scott KP, et al. Links between diet, gut microbiota composition and gut metabolism. Proc Nutr Soc 2015;74:13-22.
4. Brahe LK, Astrup A, Larsen LH. Is butyrate the link between diet, intestinal microbiota and obesity-related metabolic diseases? Obes Rev 2013;14:950-9.
5. Albillos A, de Gottardi A, Rescigno M. The gut-liver axis in liver disease: Pathophysiological basis for therapy. J Hepatol 2020;72:558-577.
6. de Cabo R, Mattson MP. Effects of Intermittent Fasting on Health, Aging, and Disease. N Engl J Med 2019;381:2541-2551.
7. Su J, Wang Y, Zhang X, et al. Remodeling of the gut microbiome during Ramadan-associated intermittent fasting. Am J Clin Nutr 2021;113:1332-1342.
8. Su J, Li F, Wang Y, et al. Investigating Ramadan Like Fasting Effects on the Gut Microbiome in BALB/c Mice. Front Nutr 2022;9:832757.

Introduction

Sarcopenia, the progressive loss of skeletal muscle mass, is related to metabolic-dysfunction associated steatotic liver disease (MASLD) and cardiovascular damage (CVD). Genetic variants like PNPLA3, TM6SF2, and HSD17B13 influence MASLD risk. The prevalence of CVD is higher in MASLD patients, however the impact of sarcopenia on CVD in MASLD patients is undefined. Moreover, the role of genetic variants in sarcopenia and CV damage is unexplored.

Aims & Methods

To assess the impact of sarcopenia, metabolic comorbidities, and genetic variants on liver and CVD in a cohort of non-cirrhotic MASLD patients.
We enrolled 841 MASLD patients. Fibrosis was assessed by liver stiffness measurement (LSM) (LSM≥8 kPa for advanced fibrosis) and steatosis by controlled attenuation parameter (CAP) at Fibroscan (CAP>280 dB/m for severe steatosis). Sarcopenia was defined by the lowest tertile of skeletal muscle index (SMI = skeletal muscle mass/height^2) at bioimpedance analysis. CV damage markers included carotid intima-media thickness (cIMT)≥0.9 mm, carotid plaques, epicardial fat thickness (EFT) ≥5.2 mm. Increased waist circumference (WC) >102/88 cm in men/women. Genetic polymorphisms in PNPLA3, TM6SF2, and HSD17B13 were assessed in 424 patients.

Results

Mean age was 51 ys, 63% male. 43% were obese, 65% had increased WC. 50% were dyslipidemic, 25% diabetic. 24% had advanced fibrosis, 71% severe steatosis. 22% had increased cIMT, 35% had carotid plaques, 83% had increased EFT. Sarcopenia (SMI<10.35/7.75 kg/m2 in men/women) was linked to older age (54 vs 48 ys, p<0.001), lower BMI (27 vs 33.4 kg/m2, p<0.001), WC (99 vs 110 cm, p<0.001), CAP (293 vs 317 dB/m, p<0.001), and LSM (4.9 vs 6.4 kPa, p<0.001) compared to non-sarcopenic. Sarcopenic also had higher dyslipidemia (56% vs 46%, p=0.03), increased cIMT (28% vs 18%, p=0.01), and EFT (85% vs 77%, p=0.05). At adjusted multivariate analysis, sarcopenia remained associated with low BMI (OR 0.61; 95% CI 0.5-0.7, p<0.001), WC (OR 0.92; 95% CI 0.8-0.97, p=0.001), female sex (OR 2.21; 95% CI 1.2-4.0, p=0.008), and increased cIMT (OR 2.1; 95% CI 1.1-3.97). In sarcopenic, PNPLA3 CG/GG variant was linked to increased LSM (OR 1.82, 95% CI 1.1-3.1, p=0.03) but lower cIMT (OR 0.39, 95% CI 0.15-0.96, p=0.04). TM6SF2 wild-type allele was associated with increased cIMT (OR 3.4, 95% CI 1.3-5.8, p=0.004), and WC (OR 2.3, 95% CI 1.4-3.6, p=0.001). No differences were seen in HSD17B13 variant.
Analyzing different metabolic subgroups, in diabetic and hypertensive patients, prevalence of carotid plaques was higher in sarcopenic vs non-sarcopenic (56% vs 30%, p=0.04, 58% vs 20%, p<0.001). In hypertensive patients sarcopenia remained an independent risk factor for carotid plaques (OR 1.9, CI 95% 1.05-3.5, p=0.03) and high/very high CV risk (OR 3.0, CI 95% 1.2-7.2, p=0.01) in multivariate analysis adjusted for age, sex, diabetes, dyslipidemia, smoke and BMI, whereas in non-diabetic subjects for high/very high CV risk (OR 1.44, CI 95% 1.1-2.1, p=0.04).

Conclusion

MASLD patients, particularly non-obese, have a high prevalence of sarcopenia and increased subclinical CVD. Despite lower visceral adiposity, in hypertensive patients sarcopenia seems to potentiate the CVD. In sarcopenic patients, the PNPLA3 variant correlated with lower subclinical atherosclerosis, while TM6SF2 polymorphism showed a protective role against CV damage.
MASLD patients would need a prompt evaluation and treatment of sarcopenia to prevent the onset of CVD, particularly if hypertensive. Combining genetic data is required to clarify the role of sarcopenia in MASLD.