Review Article

Emerging Role of the Gut Microbiome in the Progression of Nonalcoholic Fatty Liver Disease and Potential Therapeutic Implications

Saumya Jayakumar; Rohit Loomba

Aliment Pharmacol Ther. 2019;50(2):144-158.

Abstract and Introduction

Abstract

Background: Nonalcoholic fatty liver disease (NAFLD) is a prevalent disorder associated with obesity and diabetes. Few treatment options are effective for patients with NAFLD, but connections between the gut microbiome and NAFLD and NAFLD-associated conditions suggest that modulation of the gut microbiota could be a novel therapeutic option.

Aim: To examine the effect of the gut microbiota on pathophysiologic causes of NAFLD and assess the potential of microbiota-targeting therapies for NAFLD.

Methods: A PubMed search of the literature was performed; relevant articles were included.

Results: The composition of bacteria in the gastrointestinal tract can enhance fat deposition, modulate energy metabolism and alter inflammatory processes. Emerging evidence suggests a role for the gut microbiome in obesity and metabolic syndrome. NAFLD is often considered the hepatic manifestation of metabolic syndrome, and there has been tremendous progress in understanding the association of gut microbiome composition with NAFLD disease severity. We discuss the role of the gut microbiome in NAFLD pathophysiology and whether the microbiome composition can differentiate the two categories of NAFLD: nonalcoholic fatty liver (NAFL, the non-progressive form) vs nonalcoholic steatohepatitis (NASH, the progressive form). The association between gut microbiome and fibrosis progression in NAFLD is also discussed. Finally, we review whether modulation of the gut microbiome plays a role in improving treatment outcomes for patients with NAFLD.

Conclusions: Multiple pathophysiologic pathways connect the gut microbiome with the pathophysiology of NAFLD. Therefore, therapeutics that effectively target the gut microbiome may be beneficial for the treatment of patients with NAFLD.

Introduction

Nonalcoholic fatty liver disease (NAFLD) is characterised by fat accumulation (or steatosis) in >5% of hepatocytes in individuals who either consume little alcohol or have no other secondary causes of steatosis such as viral hepatitis, lipodystrophy or medications associated with the development of steatosis.^[1,2] The increasing prevalence of NAFLD parallels rises in the incidence of obesity and insulin resistance.^[3,4] NAFLD is among the most common causes of liver disease and liver transplantation in the Western hemisphere.^[4,5] NAFLD can be sub-classified into two categories: the non-progressive form, nonalcoholic fatty liver (NAFL) and the progressive form, nonalcoholic steatohepatitis (NASH).^[1,6] NASH is a clinic-pathologic entity that is typically characterised by the presence of zone 3 steatosis, ballooning and lobular inflammation; perisinusoidal fibrosis may or may not be also present.^[1,7] Fibrosis progression rate is estimated to be higher in NASH than in NAFL and progression to cirrhosis may take up to 30 years; however, rapid progression to cirrhosis may occur in a small subset of patients.^[8] In addition, NASH is associated with increased risk of hepatocellular carcinoma (HCC) and all-cause (cardiovascular and liver-related) mortality.^[9–11]

NAFLD pathogenesis is related to multiple insults that occur simultaneously and may act synergistically,^[12,13] including accumulation of triglycerides (TGs),^[12] mitochondrial dysfunction and increased oxidative stress,^[12–14] altered mechanisms of apoptosis and autophagy,^[15,16] increased levels of toxic lipid-related factors (eg, free fatty acids)^[12] and liver inflammation.^[12] Genetics (eg, mutations in the patatin-like phospholipase domain-containing 3 gene)^[17] and adverse consequences of dietary habits and sedentary lifestyle (eg, insulin resistance, central obesity, dyslipidaemia and hypertriglyceridemia) likely contribute to overall NAFLD pathophysiology and augment the underlying mechanisms of liver insult.^[12]

Accumulating evidence also implicates the gut microbiota in the development and progression of NAFLD^[18,19] and suggests that therapeutic agents that target the gut microbiota may be beneficial. This review examines the pathophysiologic implications of altered gut microbiota in NAFLD and highlights recent progress in the development of microbiota-targeting therapies for patients with liver disease.

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Table 1. Human studies of probiotics for NAFLD
Publication	Study population	Study design/treatments	Primary outcomes
Miccheli et al 2015¹³²	Obese children with elevated ALT and ultrasonographic and histologic evidence of NAFLD	DB, RCT; patients received placebo or the probiotic medical food VSL#3^a qd (1 package for patients aged < 10 y and 2 packets for patients aged > 10 y) for 4 mo	Among patients who completed the study (n = 22 for both groups), BMI, AST, total and active GLP-1 levels, and presence of fatty liver at 4 mo significantly improved in the probiotic group vs the placebo group (P < 0.05) No significant differences in TGs, cholesterol, HDL, LDL or glycometabolism indices (glucose, insulin and HOMA-IR) were observed between groups at 4 mo Key metabolites (valine, 3-aminoisobutyrate, alanine, tyrosine and pseudouridine) were significantly lower in the probiotic group vs placebo group at month 4 (P < 0.05), indicating an effect on BCAA and AAA metabolism, oxidative stress and microbiota metabolic pathways
Alisi et al 2014¹³³	Obese children (median age, 10-11 y) with histologically diagnosed NAFLD	DB, RCT; patients received placebo or VSL#3^a (1 sachet/d for patients aged < 10 y or 2 sachets/d for patients aged > 10 y) for 4 mo	At 4 mo, risk of severe steatosis was significantly lower in VSL#3 groups (n = 22) vs placebo (n = 22) (P < 0.001) BMI was significantly reduced with VSL#3 vs controls at 4 mo (P < 0.001) At 4 mo, there was a trend towards a reduction in GLP-1 in the VSL#3 group No significant changes were observed with VSL#3 vs placebo for TGs, HOMA-IR or ALT levels
Nabavi et al 2014¹³⁴	Patients with NAFLD (unspecified diagnosis)	DB, RCT; patients received 300 g/d of conventional yogurt containing L bulgaricusand S thermophilus, or yogurt enriched with B lactis Bb12 and L acidophilus La5 for 8 wk	Serum levels of ALT, AST, TC and LDL-C were reduced significantly with probiotic-enriched yogurt (n = 36) vs normal yogurt (n = 36) Probiotic-enriched yogurt significantly reduced ALT, LDL-C, AST and TC serum levels vs baseline (P < 0.05 for all)
Shavakhi et al 2013¹³⁵	Adults with histologically confirmed NASH, persistent elevation of ALT, and alcohol consumption < 20 mg in men or < 10 g in women	DB, RCT; patients received 2 tablets of metformin 500 mg and either probiotic supplement daily or placebo for 6 mo	After 6 mo of treatment, levels of ALT and AST were significantly reduced with metformin and probiotic treatment (n = 31) compared with metformin and placebo (n = 32) (P < 0.001 for each) At 6 mo, BMI, TG and TC levels were significantly reduced with metformin and probiotic treatment compared with metformin and placebo (P ≤ 0.02 for all)
Wong et al 2013¹³⁸	Adults with histology-proven NASH	OL, RCT; patients received "usual care" or 1 sachet of Lepicol^®b b.i.d. for 6 mo	At 6 mo, a significant between-group change in AST level was noted (P = 0.02) Patients in the probiotic group (n = 10) tended to have greater reductions in IHTG at 6 mo than the usual care group (n = 10) No significant alterations in TG, BMI, ALT, fasting glucose, TC, HDL-C, LDL-C, hepatic TG or liver stiffness were observed between groups at 6 mo
Aller et al 2011¹³⁶	Patients with biopsy-proven NAFLD	DB, RCT; patients received placebo or 1 tablet containing L bulgaricus and S thermophilus qd for 3 mo	After 3 mo, significant reductions from baseline in ALT, AST and GGT were observed in the probiotic group (n = 14) (P < 0.05) but not the placebo group (n = 14) Neither treatment had any effects on glucose, TC, LDL-C, HDL-C, TG, insulin, HOMA-IR, IL-6 or TNF-α levels
Vajro et al 2011¹³⁷	Paediatric patients with a BMI > 95th percentile for their age and sex who had liver abnormalities (eg, increased ALT levels) associated with ultrasound evidence of fatty liver (n = 20)	DB, RCT; patients received either Lactobacillus GG 12 billion CFU/d (n = 10) or placebo (n = 10) for 8 wk	No significant between-group differences in the number of patients who achieved ALT values < 40 U/L ALT levels and concentration of peptidoglycan-polysaccharide were significantly higher with probiotics vs placebo (P = 0.03 for each) No significant differences in BMI, visceral fat, TNF-α levels or hepatorenal ultrasonographic ratio
Loguercio et al 2005¹³⁹	Patients with biopsy-proven NAFLD (n = 22) alcoholic cirrhosis (n = 20), HCV (n = 20) or HCV-related cirrhosis (n = 16)	OL; patients received VSL#3^a for 3 mo	After 3 mo, plasma levels of AST and ALT were significantly improved in all patients (P < 0.01) vs baseline In patients with NAFLD or alcoholic cirrhosis, VSL#3 significantly reduced markers of oxidation (MDA and 4-HNE) from baseline at 3 mo (P < 0.01) Levels of S-NO were significantly reduced in all groups (P < 0.05)

Abbreviations: 4-HNE, 4-hydroynonenal; AAA, aromatic amino acids; ALT, alanine aminotransferase; AST, aspartate aminotransferase; b.i.d., twice daily; BCAA, branched chain amino acids; BMI, body mass index; CFU, colony-forming unit; CRP, C-reactive protein; DB, double-blind; GGT, gamma-glutamyl transferase; GLP-1, glucagon-like peptide 1; HCV, hepatitis C virus; HDL, high-density lipoprotein; HDL-C, high-density lipoprotein cholesterol; HOMA-IR, homeostasis model assessment of insulin resistance; IHTG, intrahepatic triglycerides; LDL, low-density lipoprotein; LDL-C, low-density lipoprotein cholesterol; MDA, malondialdehyde; NAFLD, nonalcoholic fatty liver disease; NASH, nonalcoholic steatohepatitis; OL, open label; qd, once daily; RCT, randomised controlled trial; S-NO, S-nitrosothiol; TC, total cholesterol; TG, triglyceride; TNF-α, tumour necrosis factor alpha.
^aVSL#3 (Alfasigma USA, Inc; Covington, LA, USA) is a probiotic mixture containing S thermophilus, B breve, B infantis, B longum, L acidophilus, L plantarum, L paracasei, and L delbrueckii ssp bulgaricus.
^bLepicol® (Healthy Bowels Company Ltd; Birmingham, UK at the time of the study) contains L plantarum, L delbrueckii ssp bulgaricus, L acidophilus, L rhamnosus, B bifidum, and fructooligosaccharides.

Table 2. Human studies of nonsystemic antibiotics for NAFLD
Publication	Study population	Study design/treatments	Primary outcomes
Cobbold et al 2017¹⁴⁵	Adults with histologically confirmed NAFLD	OL rifaximin 400 mg b.i.d. for 6 wk (n = 15)	No significant alterations from baseline in ALT, hepatic lipid content, hepatic insulin sensitivity or serum cytokine (TNF-α and IL-1β) after 6 wk ALT (P = 0.017), HDL (P = 0.004) and HOMA-IR (P = 0.05) levels significantly increased from baseline to week 12 (ie, during the 6-wk post-treatment period) No consistent differences were observed in faecal microbiota composition at the phylum level Significant reduction in urinary hippurate levels with rifaximin treatment (P = 0.048) was reported, indicating possible alteration in gut microbiota metabolism
Gangarapu et al 2015¹⁴⁶	Adults with histologically confirmed NAFLD (steatosis, n = 15; NASH, n = 27)	OL rifaximin 1200 mg/d for 4 wk (n = 42)	At 4 wk post-treatment, rifaximin significantly reduced levels of ALT (P = 0.01) and ferritin (P = 0.004) from baseline in patients with steatosis In patients with NASH, rifaximin significantly reduced BMI, ALT, AST, GGT, LDL and ferritin levels, plasma endotoxin concentrations and serum IL-10 levels from baseline to 4 wk post-treatment (P ≤ 0.01 for all) No changes in serum TNF-α, IL-1, IL-6, IL-12 or TLR-4 levels were observed in either patient group
Kakiyama et al 2013¹⁴⁷	Adult patients with "early" cirrhosis (Child-Pugh Class A without history of decompensation)	Longitudinal sub study; rifaximin 550 mg b.i.d. for 8 wk (n = 6)	Reduction in ratio of secondary BAs to primary BAs after rifaximin treatment No significant change in bacteria composition of the gut microbiota, except for reduction in Veillonellaceae

Abbreviations: ALT, alanine aminotransferase; AST, aspartate aminotransferase; b.i.d., twice daily; BA, bile acid; BMI, body mass index; GGT, gamma-glutamyl transferase; HDL, high-density lipoprotein; HOMA-IR, homeostasis model assessment-insulin resistance; IL, interleukin; LDL, low-density lipoprotein; NAFLD, nonalcoholic fatty liver disease; NASH, nonalcoholic steatohepatitis; OL, open label; TLR, toll-like receptor; TNF-α, tumour necrosis factor alpha.

Table 3. Animal studies of faecal microbiota transplantation for NAFLD
Publication	Study population	Study design/treatments	Primary outcomes
Nicolas et al 2017¹⁵⁶	Donors: WT mice fed a high-fat diet WT mice fed a normal calorie diet Genetically obese (ob/ob) mice Recipients: WT mice	Recipients were gavaged with gut microbiota obtained from cecum of (a) WT mice fed a normal diet; (b) WT mice fed a high-fat diet; (c) genetically obese mice	Gut microbiota from both WT mice fed a high-fat diet and genetically obese mice reduced hepatic gluconeogenesis and adiposity elicited by a high-fat diet
Li et al 2015¹⁵⁷	Donors: WT mice Recipients: WT mice that received ceftriaxone b.i.d. for 7 d to induce gut microbiota dysbiosis	Recipients gavaged for 3 d with faecal microbiota from WT mice or cultured bacteria initially isolated from donor mice faeces	Gavage with faecal microbiota from WT mice or cultured bacteria improved inflammatory cell infiltration, tissue architecture distortion and vascular congestion elicited by ceftriaxone Ceftriaxone-induced intestinal permeability was significantly improved with administration of faecal microbiota or cultured bacteria after 1 and 2 wk vs untreated animals (P < 0.05)