MASLD, the liver disease associated with metabolic dysfunction in children

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Author(s):

	Antonella Mosca
	MD, Hepatology and Liver Transplant Unit, Bambino Gesù Children’s Hospital, IRCCS, 00165 Rome, Italy.
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	Andrea Pietrobattista
	MD, Hepatology and Liver Transplant Unit, Bambino Gesù Children’s Hospital, IRCCS, 00165 Rome, Italy.
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This article is dedicated to:

	Valerio Nobili ✝︎
	“Bambino Gesù” Children Hospital, P.zzle Sant’Onofrio, 4, 00165 Rome Italy
	View Author’s Full Biography

The term nonalcoholic fatty liver disease (NAFLD) has been used to describe the histological spectrum characterized by steatosis to steatohepatitis (NASH). NAFLD is defined by infiltration of liver fat >5% hepatocytes, assessed by liver biopsy, in the absence of excessive alcohol intake (<20 g/day) and evidence of viral, autoimmune, or drug-induced liver disease. It encompasses a spectrum of liver diseases ranging from simple accumulation of intrahepatic fat (steatosis) to variable degrees of necrotic inflammation and fibrosis (steatohepatitis ) .   

Since the studies conducted have always placed NAFLD in association with other pathologies, almost always of metabolic origin, in 2020 several scientific societies proposed to change the definition and use the term fatty liver disease associated with metabolic dysfunction (MAFLD), which includes patients with fatty liver disease regardless of the amount and pattern of alcohol intake in this terminology. However, MAFLD is limited to the obese population .  For this reason, in 2023, a committee of international experts redefined hepatic steatosis, removing “fatty” from the definition. Moreover, in this Delphi consensus, the term steatohepatitis was considered an important pathophysiological concept that should be retained. The name chosen to replace NAFLD was fatty liver disease associated with metabolic dysfunction (MASLD) . The new definition closes the pathophysiological link of fatty liver with metabolic dysfunction and insulin resistance, reinforcing the role of cardiometabolic risk factors associated with MASLD.  MASLD is characterized by a broad spectrum of liver abnormalities ranging from simple steatosis to steatohepatitis associated with metabolic dysfunction (MASH) with concomitant lobular inflammation, swelling, and fibrosis. This clinical condition could degenerate into https://www.sciencedirect.com/topics/pharmacology-toxicology-and-pharmaceutical-science/fibrosiscirrhosis and possibly hepatocellular carcinoma (HCC). To date, a multitude of genetic, epigenetic, and environmental modifiers of MASLD have been reported especially in the perinatal period and in the first years of life. However, diagnosis is often delayed due to its non-specific clinical manifestation and its discovery occurs during routine examinations.  MASLD, however, opens up new scenarios at the pediatric level, about 20-30% of adult patients with MASLD develop steatohepatitis associated with metabolic dysfunction (MASH) leading to liver cirrhosis and rarely HCC .  

Fat storage and insulin resistance (IR) 

An increase in visceral adipose tissue (VAT) and a thin superficial layer of abdominal subcutaneous fat (SAT) are associated with the propensity to store fat in the liver and muscles leading to IR and metabolic syndrome (MetS). It is known that the VAT/(VAT + SAT) ratio is a predictor of changes in intrahepatic fat content, IR grade, and altered glucose metabolism. Based on this “lipodystrophy model”, poor expandability of SAT could lead to excessive lipid accumulation in VAT, which is a major source of proinflammatory cytokines and free fatty acids (FFA), the two main factors inducing the development of NAFLD and IR . 

In addition, the accumulation of triglycerides (TG), which result from the esterification of glycerol and FFA, induces the IR state. However, this state must be differentiated into systemic IR, characterized by altered blood glucose concentrations, adipose tissue IR, characterized by the inability of insulin to suppress lipolysis, and hepatic IR, in which there is impaired hepatic glucose production . The overexpression of the sterol protein-1c regulatory element (SREBP-1c) with a consequent upregulation of de novo lipogenesis, cause an impairment of the β-oxidation of FFA and ultra-low-density lipoproteins (VLDL), with a further increase in the accumulation of hepatic lipids . The overload of FFA in hepatocytes together with the release of adipokines and proinflammatory cytokines from adipose tissue cause lipotoxicity, resulting in mitochondrial dysfunction, increased production of cytotoxic reactive oxygen species (ROS), and endoplasmic reticulum stress . 

Dietary factors: fructose and sugar 

Carbohydrates can be converted into TGs, and fructose is the one most closely associated with NAFLD, compared to glucose. Fructose consumption among children, largely in the form of high-fructose corn syrup (HFCS), a mixture of fructose and glucose monosaccharides, has increased in recent years .  Diets high in sugar (sucrose and/or HFCS) are known to increase not only the risk of NAFLD, but also of NASH. In fact, fructose intake from added sugars in processed foods is related to the epidemic increase in obesity, MetS, and NAFLD . Fructose-induced hepatic fat accumulation involves the stress pathway which results in gluconeogenesis, an increase in fat synthesis, and a decrease in fat oxidation . Fructose can modulate lipogenic enzymes by increasing the expression of sterol-1c regulatory element-binding protein (SREBP-1c) and carbohydrate-sensitive element-binding protein (ChREBP) . Chronic fructose consumption induces leptin resistance, accelerating obesity induced by high fat content .  

Inflammatory pathways and oxidative stress 

Patients with NAFLD have elevated levels of oxygen free radicals (ROS), lipid peroxidation products, and reduced concentrations of antioxidant enzymes such as superoxide dismutase (SOD) .  In NAFLD, oxidative stress induces β-oxidation of fatty acids, which promotes ROS production leading to oxidative damage of the mitochondrial membrane . This further worsens the oxidative damage that leads to hepatocellular death and progression of NASH. This inhibition amplifies cytotoxic ROS and lipid peroxidation. These products can diffuse into the extracellular space, affect Kupffer cells and stellate liver cells (HSCs), and activate the nuclear factor-κβ (NF-κβ) pathway, which results in the subsequent synthesis of TNF-α and many other proinflammatory and fibrogenic cytokines .  

Gut-liver axis  

The gut barrier and gut microbiota appear to play a key role in liver damage and its progression. Under normal conditions, only a small number of bacterial products enter the liver through the portal circulation. However, bacterial dysbiosis or alterations in the gut barrier will increase bacterial flow in the liver, thereby stimulating inflammation through Toll-Like Receptors (TLR) signaling and activation of other pattern recognition receptors in the Kupffer cells . The gut microbiota also plays a critical role in maintaining the integrity of the gut barrier and leaky gut. Dysbiosis can damage the intestinal epithelium and destroy tight junction proteins, which are important to prevent harmful substances from the gut such as bacteria, ethanol, and endotoxins enter the portal blood .  

Genetic factors 

Genetic factors are also important in the development of MASLD. Genes are involved in inflammation, lipid metabolism, and oxidation and are associated with progressive liver disease, IR, type 2 diabetes mellitus, and a higher risk of hepatocellular carcinoma and MetS.  PNPLA3 is the most documented MASLD-related gene .  The PNPLA3-I148M polymorphism increases the risk of MASLD, but without a strong effect on the components of MetS, while abdominal fat may drive the effect of this polymorphism on liver damage. In obese children, weight loss can weaken the effect of this polymorphism . Many other genetic polymorphisms have been identified that appear to be related to the pathogenesis of MASLD, while others interact with age, insulin resistance, and body mass index, and increase the risk of steatosis  (Figure 1).  

Figure 1: MASLD Development.  

MASLD development is correlated to mechanisms contributing to hepatic insulin resistance, increased VLDL synthesis, and increased hepatic glucose production. Long-chain fatty acids (LCFAs), proinflammatory cytokines, and decreased adiponectin from expanded and inflamed visceral adipose tissue subjected to lipolysis lead to an increase in hepatic synthesis of various lipid species, including ceramide, di-palmitoylphosphatidic acid (di-P PA), diacylglycerol (DAG). These act promoting hepatic insulin resistance, liver inflammation, and lipid blood cell accumulation. With lipid globule remodelling and the synthesis of phospholipids, cholesterol esters, and apolipoproteins, VLDL particles are assembled for export from the liver. With NAFLD, particularly in the absence of genetic polymorphisms known to be associated with increased lipid cell accumulation (such as the polymorphism of PNPLA3 gene), there is an increase in VLDL production, with in turn increase plasma TGs concentrations. 

Diagnosis of MASLD 

NAFLD is the most common liver disease in children, but the main problem in the diagnostic approach to the disease is the paucity of suggestive clinical signs. In fact, the diagnosis of NAFLD is often made following the occasional finding of hypertransaminasemia.  There is a lack of uniformity in the literature on which screening tool is most effective for identifying the disease in at-risk individuals. The European Society of Paediatric Gastroenterology, Hepatology and Nutrition (ESPGHAN) suggests using both serum ALT concentration measurement and liver ultrasound (US) as a screening method; the North American Society of Pediatric Gastroenterology Hepatology and Nutrition (NASPGHAN) indicates to use ALT and not US. The National Institute for Health Care (NICE) suggests using US and not ALT. Regardless of the method used, the finding of ALT> 30-40 IU/L or a US ≥2 score guarantees a specificity of ≥90% but the sensitivity of these tests, individually, does not exceed 50%, with an inherent risk of false negatives. Screening should be initiated between 9 and 11 years of age in children affected by obesity or overweight and with a cardiometabolic risk factor and repeated every 2 to 3 years if risk factors remain unchanged .  

Over the past 20 years, non-invasive biomarkers have been developed for the detection of steatosis and fibrosis in patients with NAFLD. In clinical practice, the NAFLD Fibrosis Score (NFS), Fibrosis Index-4 (FIB-4), AST Platelet Ratio Index (APRI), and Fibrosis Index-4 (FIB-4), work well to rule out advanced fibrosis-cirrhosis and, therefore, could be used as a first-line classification to identify patients at risk for advanced fibrosis. APRI and FIB-4 scores can help identify significant fibrosis in pediatric patients with MASLD, but they do not have positive predicting values high enough to be considered a diagnostic tool. Therefore, they cannot be considered for the diagnosis of fibrosis or for monitoring its progression in children. It is therefore necessary to continue research and development of new markers of exclusive fibrosis, which could significantly reduce the use of liver biopsy which is not entirely free of complications in children (Figure 2). 

Figure 2: flow-chart diagnosis of MASLD in children 

Imaging Tests  

Because of the asymptomatic features of NAFLD, fatty liver disease is often diagnosed incidentally via imaging checks such as abdominal ultrasound or magnetic resonance imaging (MRI). The most common imaging method for diagnosis is abdominal ultrasound, which is easily accessible and can demonstrate fat infiltration into the liver. However, when steatosis is less than 30%, sensitivity is significantly reduced. An alternative diagnostic method is magnetic resonance imaging, which is highly sensitive for small amounts of isolated steatosis. Magnetic resonance spectroscopy (MRS) measures proton signals as a function of their resonant frequency to separate the signal fractions of fat and water. MRS can detect small amounts of liver fat and is considered the most accurate non-invasive method of quantifying liver fat, but this modality is not readily available or affordable .
More recently, ultrasound-based measurements of liver stiffness can be integrated by conventional ultrasound devices such as acoustic resonance forced pulse imaging (ARFI) and shear wave elastography (SWE) or obtained via a dedicated device, most commonly Vibration-controlled Transient Elastography (VCTE), commercially available as the FibroScan®. ARFI and SWE use high-frequency ultrasonic pulses to generate fine waves and require the operator to define a region of interest and obtain a series of liver stiffness measurements . Limited studies of SWE and ARFI in patients with NAFLD have demonstrated excellent diagnostic accuracy for advanced fibrosis.
Liver biopsy (LB) is considered the gold standard for diagnosing NAFLD as it is the only method that can distinguish NAFLD from NASH and provide a reliable scoring system designed to estimate disease severity. In addition, it can aid in the work of differential diagnosis and in detecting coexisting liver diseases .  LB has some limitations: 1) it is an invasive technique with risks of minor and major complications; 2) pain and bleeding are the most common complications (84% and 2.8%, respectively); 3) Other reported complications are: infections, visceral perforation, arteriovenous fistula, pneumothorax, hemothorax and death (0.6%), but to date biopsy remains the “imperfect reference standard” (Figure 3). 

Figure 3. Major histological features of paediatric NAFLD/NASH.  

Steatosis is evident in (a) (40× magnification) and (b) (10× magnification); ballooning and lipogranulomas are present in (c) and (d), respectively (40× magnification); normal liver histology, for comparison purposes (e). 

Therapy 

Diet and physical activity: Improvements in diet and increased physical activity, understood as lifestyle modifications, are currently the primary treatment for pediatric NAFLD due to the strong association with overweight and obesity. This concept has also been included in recommendations for the diagnosis and treatment of pediatric NAFLD published by the North American Society of Pediatric Gastroenterology, Hepatology and Nutrition (NASPGHAN) . A healthier diet with avoidance of sugary drinks, increasing physical activity from moderate to high intensity, and limiting screen activities to <2 hours per day are the main cornerstone for both prevention and treatment of pediatric MASLD. A small reduction in weight (weight loss of 3%-5%) may reduce fatty liver disease, but a greater degree of weight loss (no less than 7%) is needed to improve hepatic steatohepatitis.  

Insulin sensitizers: Insulin-sensitizing treatment has been considered for the management of NAFLD considering the strong association between insulin resistance and NAFLD .  One of such compounds is metformin, which can lower blood sugar by decreasing gluconeogenesis in the liver, stimulate glucose uptake in muscle, and increase fatty acid oxidation in adipose tissue . This compound has been shown to improve steatosis. However, several studies have shown conflicting data on its efficacy in down-regulating or improving the hepatic histology of ALT. The NASH Clinical Research Network conducted a multicenter, randomized, double-blind, placebo-controlled study evaluating vitamin E or metformin for the treatment of NAFLD in children (TONIC) . However, the results showed no significant changes in both ALT levels and liver histology (NAFLD Activity Score – NAS) between the groups . Thiazolidinediones, such as pioglitazone and rosiglitazone, are an additional class of insulin sensitizers that increase the storage capacity of adipocytes through proliferator-activated gamma peroxisome (PPAR-γ) receptors in white adipose tissue . Several studies have demonstrated the positive effect of pioglitazone on improving steatosis and lobular inflammation in adult patients with NASH, but this class of drugs has not been authorized for pediatric age due to its cardiovascular consequences . 

Omega-3 fatty acids – Docosahexaenoic acid (DHA): Omega-3 fatty acids are a variety of polyunsaturated fatty acids (PUFAs). The most studied omega-3s in humans are eicosapentaenoic acid (EPA) and DHA, found in fish, algae, and fish oil. Omega-3s are modulators of the transcription of genes that regulate lipid metabolism and provide systemic anti-inflammatory and insulin-sensitizing activity.  DHA shows an anti-inflammatory effect by activating through GPR120 (G protein-coupled receptor) and PPAR-γ. Both receptors control inflammatory signaling. DHA activation has several effects: 
1) it inhibits the T proliferation of macrophages and lymphocytes (decreasing the production of IL-2) ; 
2) it reduces NFκβ activation in response to endotoxin, likely acting on MyD88;
3). PPAR-γ pathway (along with NFκβ inactivation) reduces the production of inflammatory cytokines TNF, IL-6 and IL-1β .  

In a randomized controlled trial, children with NAFLD on an 18-month regimen of daily DHA had histologically proven improvement in fatty liver disease, ballooning, inflammation, NAS and PNHS (Pediatric NAFLD Histological Score) scores, and a reduction in ductular reaction and hepatic stem/progenitor cells (HSC), as well as an improvement in laboratory biomarkers such as transaminases, insulin, and HOMA-IR. However, no effect on fibrosis was observed .   

DHA and Vitamin D (VD): In recent decades, many studies have emphasized the usefulness of a therapeutic approach with VD and DHA. VD receptor (VDR) ligands have been shown to inhibit HSC TGFb1/SMAD(small mother against decapentaplegic protein) activation and that DHA binding to GPR120 has an anti-inflammatory effect .  After TGFb1 activation, SMAD translocation in the nucleus activates HSCs in response to liver injury. This pathway acts to regulate hepatic fibrogenesis. Activation of VDR, a hormone nuclear receptor whose endogenous activators are the biologically active form of VD and bile acids, may antagonize the SMAD effect. There is a lot of evidence that VD has a beneficial effect on fibrogenesis, improving fibrosis score, especially in children, although it does not affect lipid profile, markers of insulin resistance, and inflammation as well as an improvement in fibrosis score and reduced HSC activation .  

Probiotics.  NAFLD is associated with increased intestinal permeability, intestinal bacterial overgrowth, and alterations in the composition of the gut microbiota . Therefore, several authors have suggested modulation of the gut microbiota by probiotics as a possible approach for obesity and NAFLD . Nobili V. et al showed an improvement in steatosis in children with biopsy-proven MAFLD after 4 months of treatment with a mixture of eight probiotic strains (Streptococcus thermophilus, bifidobacteria , Lactobacillus acidophilus, L. lantarum, L. paracasei, and L.  bulgaricus). The authors observed a significant reduction in BMI and histological parameters of MAFLD and MASH, as well as an increase in circulating levels of GLP-1 and its activated form (aGLP-1) in patients treated . In addition, the authors observed that Lactobacillus mucosae was significantly more present in obese MAFLD and MASH children, while Bifidobacterium spp. was more abundant in control subjects, suggesting a protective role of these microorganisms against both obesity and the development of MAFLD . 

Bariatric surgery can be considered for adolescents with severe obesity (BMI > 40 kg/m² with severe comorbidities or with BMI > 50 kg/m² with mild comorbidities), with NASH and significant fibrosis (ISHAKscore≥1).  Roux-en-Y gastric bypass (RYGB) and Laparoscopic Adjustable Gastric Banding (LAGB) were the 2 main surgical procedures that have been used in pediatric obesity. RYGB is considered a safe and effective option for adolescents with extreme obesity, as long as appropriate long-term follow-up is provided . In last decade the sleeve gastrectomy, as well as other types of weight loss surgery, has been used in adolescent. Several studies showed the significant reduction of BMI, but also the histologic improvement after laparoscopic sleeve gastrectomy (LSG).  These pictures are associated with the reduced activation of local cellular compartments (hepatic progenitor cells, HSCs, and macrophages), thus, strengthening the role of cellular interactions and hepatic adipocytokine production in the pathogenesis of NAFLD .  

New Treatments 

The increase in the prevalence of NAFLD, associated with the absence of effective pharmacological treatments, has led to the study of new molecules to counteract NAFLD (Table 1). 

Table 1. 

Source: www.clinicaltrials.gov  

Olive oil, with high amounts of monounsaturated fatty acids (MUFAs) and antioxidant phenolic compounds, is considered one of the main constituents of the Mediterranean diet. It is claimed that olive oil may reduce TG accumulation in the liver by improving plasma TG concentration in the postprandial by interacting with glucose-1 and glucagon-1 peptide responses in patients with insulin resistance . These effects are thought to be mediated by several mechanisms, including reducing the activation of nuclear factor κ, reducing LDL oxidation, and improving IR by reducing the production of pro-inflammatory cytokines (TNF-α and IL-6). Hydroxytyrosol (HXT) is classified as a phytochemical compound that expresses very strong antioxidant properties. In nature, HXT is found in high concentrations in olives. The HXT molecule is one of the naturally occurring polyphenolic compounds with increased antioxidant activity .  Treatment with HXT and Vit E appears to reduce NAFLD-related systemic inflammation in children, primarily through an increase in circulating IL-10 levels that occurred in response to DNA damage recovery, ultimately improving steatosis and hypertriglyceridemia .  

Farnesoid X receptor (FXR) is a nuclear receptor, expressed primarily in the liver and intestine, that binds to bile acids. When activated, FXR migrates to the cell nucleus and modulates the transcription of specific genes involved in the regulation of inflammation and glucose and lipid metabolism. Several studies, mostly based on animal models, have shown that the use of FXR-agonists (e.g., obeticholic acid) could improve hepatic steatosis and steatohepatitis . 

Glucagon-like peptide 1 (GLP-1) analogues, i.e. liraglutide and semaglutide, incretins that are analogues of the gut-derived hormone GLP1, can induces weight loss and insulin sensitivity, and appear to play a role in reducing fatty liver disease as well as inflammation and progression of damage . 

In conclusion, the current ideal pharmacological approach for the treatment of NAFLD is represented by a “combination therapy” consisting of a mix of available agents, with the aim of blocking and regressing liver damage in treated patients. The ideal treatment should be administered in a personalized way on the patient, based on the medical evaluation considering the different histological characteristics of MASLD and the main metabolic alterations, in order to combine the different therapeutic effects to achieve the best result.  

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