Non-Alcoholic Fatty Liver in Children with Obesity: Identifying Risk Factors and Management Strategies

With increasing rates of childhood obesity, nonalcoholic fatty liver disease (NAFLD) has become the most common liver disease among children worldwide [1]. The prevalence of NAFLD in Chinese children was 3.4% [2]. The prevalence in obese and overweight children was significantly higher, ranging between 50 and 80% [3].

NAFLD can progress from simple steatosis (SS) to non-alcoholic steatohepatitis (NASH), which subsequently leads to fibrosis/cirrhosis [4, 5]. Among obese youth with NAFLD, 10% have NASH, which is characterized by inflammation and expansion of hepatocytes in the setting of hepatic steatosis [1, 6–8].

Although simple hepatic steatosis usually has a "benign course," NASH can progress to end-stage liver disease. Children can reach a harmful stage faster than adults [9]. Liver cirrhosis due to NAFLD has been described in children [10].

In addition to intrahepatic lesions, NAFLD also has serious health consequences beyond the liver, associated with metabolic disorder, cardiovascular disease, and insulin resistance [11, 12].

Among patients with NASH, half of the deaths were due to cardiovascular diseases and malignancy [13, 14]. Therefore, early identification of SSc and NASH is crucial for treatment and prognosis.

Liver biopsy is considered the gold standard for the diagnosis of NAFLD, which can facilitate the differentiation of SSc and NASH in both children and adults [15]. However, it is not suitable for screening children due to its invasive nature and cost. Additionally, only a small portion of the liver is examined, which can lead to sampling errors and selection biases.

A qualitative and non-invasive alternative is to examine liver steatosis by ultrasound [15]. Additionally, blood alanine transaminase (ALT) is an inexpensive, minimally invasive, acceptable, and universally available blood test.

NAFLD is the most common cause of elevated liver enzymes in patients in developed countries. NASH is associated with a twofold increase in ALT in children with obesity [15, 16].

Studies in adults have demonstrated the link between NAFLD and metabolic disorders [17]. Longitudinal studies in adults demonstrate that patients with NAFLD have a higher incidence of diabetes, metabolic syndrome, and mortality compared to matched control populations [18]. However, studies on the associations between epidemiological situations, risk factors and cardiovascular risk of NAFLD in obese children are limited, especially in China.

Children are in a period of rapid growth and development, and their pathophysiological changes are different from those of adults. Today’s obese children are also exposed to maternal obesity and insulin resistance earlier than decades ago.

Like other liver diseases, NAFLD is asymptomatic in the initial stage and may go unnoticed by doctors. This clinical study aims to investigate the possible risk factors for NAFLD in children with obesity in the Chinese population, specifically in the city of Nanjing.

> Subjects and inclusion criteria

This study was conducted in Nanjing, China, at the Children’s Hospital of Nanjing Medical University. The study protocol was approved by the Medical Ethics Committee of Nanjing Medical University Children’s Hospital (201412004.1).

Children aged 6 to 16 years were recruited for the study from September 2015 to April 2018. The study was conducted on children with obesity. The inclusion criterion was body mass index (BMI) ≥ 95th percentile according to the BMI classification of Chinese children.

Exclusion criteria were any other endocrine disease, viral hepatitis, hereditary diseases, and other infectious diseases or chronic diseases.

The pubertal stage was evaluated based on the Tanner scale. Children with obesity were further divided into three subgroups by combining ultrasound and 2-fold ALT elevation: simple obesity (OS) group with normal liver on ultrasound, simple steatosis (SS) group with fatty liver on ultrasound and ALT <80. U/L, and NASH group with fatty liver on ultrasound and ALT ≥80 U/L, according to the laboratory double value reference standard for liver function [15].

Study staff provided oral and written information to parents/caregivers. Parents or caregivers of all subjects provided written informed consent prior to inclusion in the study.

Written consent was obtained from all children. Through interviews with the children and their parents, experienced interviewers collected basic information about the children, including birth history, family history, and lifestyles.

All participants had anthropometric evaluations. Height and weight were measured by experienced researchers using standardized measurement methods [19]. A digital scale (graduation 100 g) was used to measure weight, and a caliper (graduation 1 mm) (Seca 704, Germany) was used to measure height.

BMI was calculated by dividing body weight (in kilograms) by the square of height (in meters). The BMI standard deviation score (BMI-SD) was calculated according to the WHO reference values. End-expiratory waist circumference was measured at the umbilicus, with a soft non-retractable ruler to the nearest 0.1 cm [20].

Waist circumference (in centimeters) was divided by height (in centimeters) to calculate waist-to-height ratio (WHR).

Body fat and skeletal muscle were measured with InBody J20 (Biospace, Korea). Body fat percentage and skeletal muscle percentage were calculated. After a quiet rest for 10 minutes, blood pressure was measured three times with an electronic sphygmomanometer (Omron HBP-1300) and the mean value was taken.

All subjects were told to fast overnight for 12 h before blood collection the next morning. Fasting laboratory analyzes included glucose, insulin, total cholesterol, high-density lipoprotein cholesterol (HDL), triglycerides, ALT, and aspartate aminotransferase (AST).

All subjects were examined after 12 h of overnight fasting. Liver ultrasound was performed by an ultrasound technician to establish a diagnosis according to at least two of the following criteria: greater echogenicity in the liver than in the spleen or kidney; obliterated hepatic vasculature; high signal attenuation [21].

The diagnosis of metabolic syndrome was defined as having at least three criteria with the following cut-off points [22]: abdominal obesity (waist circumference ≥ 90th percentile of the waist circumference of children of the same age and sex [23 ], elevated triglycerides (≥1.47 mmol/L [130 mg/dL]) [24], low HDL cholesterol (<1.03 mmol/L [40 mg/dL]), elevated blood pressure [systolic blood pressure ( SBP) ≥ 90th percentile of SBP or diastolic blood pressure (DBP) ≥ 90th percentile of DBP of children of the same age, height and sex] and impaired fasting glucose (≥ 5.6 mmol/L [100 mg/L dL]).

In addition to metabolic syndrome factors, insulin resistance was defined according to the homeostasis model assessment of insulin resistance (HOMA-IR) which was calculated as fasting insulin (mU/mL) × [glucose fasting (mmol/L)/22.5] [25].

The insulin resistance threshold was defined as ≥3.16, as described in the literature [26]. Hyperuricemia was defined as a uric acid value ≥357 umol/L [27].

All statistical analysis was performed using SPSS Software version 25.0 (SPSS Inc., Chicago, Illinois). Quantitative data with normal distribution were expressed as mean ± SD. One-way ANOVA was used to test differences between the three groups, and multiple tests were analyzed with the least significant difference method.

Quantitative data with abnormal distribution were expressed as median with interquartile range. The Kruskal-Wallis test was used to test the differences between the three groups, and the Mann-Whitney U test was used for comparison between groups.

Chi-square tests were performed for differences in proportions, and the odds ratio (OR) value and 95% confidence interval (CI) were calculated. Dichotomous logistic regression analysis was used to test the risk factors for NASH, and the adjusted OR and 95% CI were provided. The level of significance was defined as p <0.05.

Demographic and clinical characteristics of the participants

A total of 428 children with obesity were recruited for the study, including 148 (34.6%) with OS, 235 (54.9%) with SS, and 45 (10.5%) with NASH. There were no significant differences in age classification or Tanner stage between the three groups.

Obesity indicators such as BMI, BMI-SD, waist circumference and body fat in the NASH group were significantly higher than those in ES and OS, but the percentage of skeletal muscle in the NASH group was significantly lower than that in the ES and OS group. in the other groups (p <0.001).

The percentage of body fat and RCA in the ES and NASH groups was significantly higher than in the OS group, but there was no significant difference between the first two groups, indicating that these two anthropometric measures were not able to distinguish well the spectrum of NAFLD.

Children in the NASH group had higher liver enzymes, including ALT and AST, indicating liver damage. Triglyceride levels were significantly higher in the NASH and ES groups compared to the OS group. The uric acid concentration and HOMA-IR levels involved in the pathogenesis of NAFLD were classified as follows: OS < ES < NAFLD.

The rates of occurrence of metabolic syndrome in the NASH and SS groups were significantly higher than in the OS group (53.3, 49.8% vs 19.6%, p < 0.001).

Children with NASH had significantly elevated blood pressure and triglycerides compared with children in the OS group, but there was no difference in the frequency of abdominal obesity, low HDL, and impaired fasting glucose when dichotomous cutoffs were used between groups.

Subsequent investigations indicated that the prevalence of insulin resistance (38.5% / 46.8% / 68.9%) and hyperuricemia (45.9% / 68.5% / 86.7%) were consistently higher from the OS group to the ES group to the NASH group, respectively (p <0.001).

NAFLD progression was positively associated with cardiovascular risk factors and metabolic syndrome in these children with obesity. All children in the SSc and NASH groups had at least one metabolic syndrome factor.

Furthermore, the distribution of metabolic syndrome characteristics in children in the NASH and ES groups was significantly (p < 0.001) shifted to the right, with more characteristics present than in obese children in the OS group.

After adjusting for all other variables among children with obesity, the odds of having NASH were significantly higher in those with severe obesity (BMI-SD ≥ 3) (OR 2.56; 95% CI 1.06–6.17) compared to those with mild obesity, men (OR 4.94; 95% CI: 1.41-17.35) compared to women, those with hyperuricemia (OR 2.98; 95% CI 1.14-7, 76) compared to those without hyperuricemia, and those with insulin resistance (OR 2.71; 95% CI 1.29– 5.69) compared to those without insulin resistance.

> Clinical characteristics of NAFLD in children with obesity

Against the backdrop of China’s childhood obesity epidemic, the rate of NAFLD has become increasingly serious over the past 20 years. In this study of 428 children with obesity, 54.9% had NAFLD and 10.5% had NASH. Additionally, children with NAFLD had a higher risk of cardiovascular and metabolic syndrome than those without NAFLD.

Severe obesity, gender, hyperuricemia, and insulin resistance are risk factors for NASH in children with obesity. Clinical evidence indicates that children with NAFLD experience increased incidence and mortality of cardiovascular diseases in adulthood [9]. Therefore, early identification of clinical features is particularly important for Chinese children with NAFLD and NASH in light of the current obesity epidemic.

Due to differences in diagnostic criteria, population selection, and ethnicity, the reported prevalence of NAFLD varies greatly in the literature [3]. A meta-analysis of epidemiological studies in children showed that the average prevalence of NAFLD was 7.6% in general population studies and 34.2% in studies of children with obesity [3].

Due to the limitations of invasive diagnostic methods for NASH, population-based studies on NASH are currently lacking. A recent community-based study reported that 193 subjects (55%) were diagnosed with NAFLD, and 105 subjects (30%) were diagnosed with NASH by biopsy [16].

In the authors’ study, the incidence of NAFLD in children with obesity was 65.4%, which was higher than other reports, and the incidence of NASH was 10.5%. The risk of NASH in severely obese children (BMI-SD ≥ 3) was 2.56 times higher than in mildly obese children (2 ≤ BMI-SD <3). These results highlight that obesity remains an important trigger of NAFLD, although NAFLD and NASH can be found in the normal BMI population.

At the same time, the authors’ results showed that the incidence of NASH in men was significantly higher than in women (14% vs. 2.4%), and gender remained a risk factor for NASH after adjusting for age. , obesity and other components of the metabolic syndrome (OR = 4.94, 95% CI (1.41–17.35)).

The gender difference in the distribution of NASH is similar to the results in adults. However, the reasons for this are unclear so far. It is possible that the distribution of fat is more likely to accumulate in the viscera of males, which is more likely to cause metabolic disorders compared with subcutaneous fat, and that estrogen may improve the tissue’s insulin sensitivity. adipose in women [28].

In the present study, children with NAFLD showed higher cardiovascular risk and more metabolic syndromes than obese children without NAFLD. This result is consistent with that reported in a previous case-control analysis [11].

Children with NAFLD were more likely to develop abnormal glucose, insulin, triglycerides, and HDL cholesterol than children without NAFLD with the same age, sex, and BMI [11]. A study including 400 children with obesity also revealed that abnormal liver ultrasound was associated with systolic and diastolic dysfunction [29].

Metabolic syndrome is a collection of cardiovascular risk factors that may predict diabetes and cardiovascular disease better than any individual component. More and more evidence has shown that a large proportion of children with NAFLD meet the diagnostic standard of metabolic syndrome, suggesting that these children will develop diabetes and cardiovascular diseases in the future.

This belief was supported by a study of a 14-year Swedish cohort of patients with NAFLD diagnosed by biopsies. There they had a diabetes prevalence of 9% at the start of the study, and the majority of patients (78%) had developed glucose intolerance or diabetes by the end of the 14-year period [18].

In the same study, survival of adults with NAFLD diagnosed by biopsy was lower than that of a matched control group, largely due to higher cardiovascular mortality [18].

Therefore, healthcare workers should pay more attention to the early identification of cardiovascular risk factors in the NAFLD population, which is useful to facilitate targeted prevention and treatment strategies.

The authors found that the risk of insulin resistance in the NASH group was significantly higher than that in the ES and OS groups, and insulin resistance was a risk factor for NASH after adjusting for confounding factors. Many clinical studies suggest that insulin resistance is always associated with NAFLD in adults and children [30-33].

A study of biopsy-proven NAFLD showed that 95% of children had insulin resistance [34]. In an experimental study, Bugianesi and colleagues found that adipose tissue is an important site for the development of insulin resistance, which may contribute to NAFLD [35]. However, the association between insulin resistance and NASH remains unclear.

The pathogenesis of NAFLD is hepatocyte steatosis and hepatocyte injury, in which insulin resistance plays an important role in the development of NAFLD.

As peripheral insulin resistance is aroused in patients with NAFLD, serum free fatty acids, synthesized through lipolysis of visceral fatty tissue and dietary fat, serve as the source of hepatic triglycerides. Insulin resistance acts on metabolic disorders induced by free fatty acids and mitochondria.

Lipid overload can also repress oxidation processes. Meanwhile, with the production of reactive oxygen species, cytokines, chemoattracted inflammatory cells are induced, and hepatic stellate cells are activated, all causing damage to hepatocytes [36].

Therefore, insulin resistance is involved in the occurrence and development of NAFLD and NASH. Because of the central role of insulin resistance in glucose metabolism, young people diagnosed with fatty liver need additional evaluation to exclude diabetes or glucose intolerance due to insulin resistance.

An increasing number of clinical studies have shown that hyperuricemia is related to NAFLD in both adults and children [37–39]. In a recent study of adolescents, hyperuricemia independently predicted [OR 2.5, 95% CI (1.87–2.83)] the presence of NASH after adjustment for potential confounders [40].

A biopsy-proven NAFLD study also found that the higher the serum uric acid, the greater the degree of lobular steatosis and inflammation [41]. As in previous studies, they confirmed that obese patients with hyperuricemia had a higher risk of NASH development [OR 2.98, 95% CI (1.14–7.76)]. Experimental studies observed that incubation of HepG2 cells with uric acid can increase intracellular triglycerides.

Possible mechanisms may be that uric acid activates mitochondrial stress, then activates endoplasmic reticulum stress, and activates sterol regulatory element binding protein 1C, while uric acid stimulates the pyrin domain of the ACE-like receptor family. NOD containing 3 inflammasomes to regulate hepatic steatosis and inflammatory response in patients with NAFLD [42, 43].

First, the authors cannot confirm that patients with suspected NASH have abnormal hepatology, so they chose the combination of ultrasound and twice the upper limit of ALT to define NASH, in order to reduce diagnostic error.

Secondly, a total of 428 children with obesity were included in this study, almost 70% were boys.

Third, due to its cross-sectional nature, the causal relationship between NAFLD and cardiovascular risk factors was not fully explored. Furthermore, other potential factors such as diet, lifestyle, and familial genetic factors should be taken into account in future studies.

In this study, the authors investigated the clinical characteristics of NAFLD and NASH in children with obesity, as well as the associations between NAFLD and metabolic risk factors in a single center.

The findings indicated that the prevalence of NASH in children with obesity is associated with high BMI-SD, gender, insulin resistance, and hyperuricemia. These findings highlight the need for early monitoring and prevention strategies for children with obesity.

More prospective, multicenter epidemiological studies are needed to determine the prevalence and development of NAFLD in children with obesity in China.