Metabolic Syndrome and Its Link to Non-Alcoholic Steatohepatitis (NASH): The Importance of Early Diagnosis

Introduction

Metabolic syndrome (MetS), often referred to as insulin resistance syndrome, presents a complex array of metabolic abnormalities that pose a significant threat to global public health. Characterized by a cluster of interrelated risk factors—such as dyslipidemia, elevated blood pressure, and insulin resistance—MetS substantially heightens the likelihood of serious health complications, including coronary heart disease, cardiovascular disease, and type 2 diabetes mellitus [1]. Central to this syndrome is abdominal obesity, which plays a pivotal role in the development of insulin resistance and associated metabolic disturbances. Recent research has broadened the scope of MetS to include chronic inflammatory and thrombotic states, non-alcoholic fatty liver disease (NAFLD), and obstructive sleep apnea, reflecting the intricate nature of these interconnections.

Globally, the prevalence of MetS is rising almost concurrently with that of obesity. As per the National Health and Nutrition Examination Survey (NHNES), the percentage of persons with MetS rose from 25.3% to 34.2% in 2012 [56]. MetS is no longer an illness that just affects adults; children and adolescents have also been documented to be affected. MetS affected 3% of children and 5% of adolescents worldwide in 2020. The prevalence of MetS increases with age; by the sixth decade of life, around 40% of individuals have MetS [57]. In some ethnic groups, MetS is significantly more common in women than in men, despite the fact that it affects both sexes equally.

NAFLD is an umbrella term that spans simple deposition of adipose tissue in the liver to more progressive steatosis with associated hepatitis, cirrhosis, fibrosis, and, in worst cases, hepatocellular carcinoma (HCC) [58]. For the sake of terminology, NAFLD is defined as non-alcoholic fatty liver (NAFL) and non-alcoholic steatohepatitis (NASH) [58]. NAFL is distinguished by steatosis of the liver involving more than 5% of the parenchyma and no signs of hepatocyte injury [59]. NASH, on the other hand, is described histologically as a necroinflammatory process in which liver cells are damaged in the presence of steatosis [59]. In terms of epidemiology, various studies have attempted to determine the real global prevalence of NAFL/NASH; however, due to severe variability in research parameters and accessible testing, no clear and trustworthy occurrence rate is presently known [58]. Having said that, estimations imply that the incidence of NAFLD is between 20% and 30% in Western nations and 5% to 18% in Asia [58]. Given current trends in dietary inattention and the predominance of sedentary lifestyles, it is not surprising that the incidence of NAFLD is increasing globally with each passing year [58].

As previously stated, NAFLD is a single entity that contains both NAFL and NASH; the distinction between them is the presence of varying degrees of inflammation and architectural remodeling, such as fibrosis. Rapid diagnosis of persons with severe fibrosis is critical for clinical care since they are more likely to develop life-threatening complications, like HCC or oesophageal varices. To diagnose NAFL, hepatic steatosis must be confirmed by imaging or histology; however, diagnosing NASH needs a liver biopsy, which is still the gold standard for describing liver histological abnormalities [3]. NAFL progresses to NASH when histology samples show evidence of hepatocellular injury, such as lobular inflammation (mixture of CD4-(+), CD8-(+) lymphocytes, Kupffer cell aggregates, polymorphonuclear leukocytes, macrophages, and T-cells), hepatocellular ballooning degeneration, apoptotic bodies, and Mallory-Denk bodies (eosinophilic intracytoplasmic inclusion composed of misfolded filaments of keratins, heat-shock proteins) [60]. The diagnosis might be difficult at times because the features of steatohepatitis do not appear evenly in all biopsies.

This article explores the relationship between MetS and NASH, highlighting the importance of timely identification and intervention to reduce long-term health burdens and associated healthcare costs.

Definition of MetS

Reaven was the first to propose the idea of "syndrome X" (later called MetS), assuming that it was a key component in the development of DMT2 and CHD, mostly via target tissue resistance to insulin action [61]. Since then, several international organizations and expert groups have made an effort to include all of the various MetS definition characteristics.

A World Health Organization advisory panel created the first formal definition in 1998. MetS is diagnosed by the WHO if insulin resistance is present, as shown by a fasting glucose level > 6.1 mmol/L (> 110 mg/dL) or a 2-hour glucose level > 7.8 mmol/L (> 140 mg/dL), as well as at least two other risk factors. Risk factors for heart disease include low HDL cholesterol (below 0.9 mmol/L or 35 mg/dL in males and below 1.0 mmol/L or 40 mg/dL in females), high triglycerides (above 1.7 mmol/L or 150 mg/dL), abdominal obesity (waist/hip ratio > 0.9 in males or > 0.85 in females, or body mass index > 30 kg/m²), and high blood pressure (systolic > 140 mmHg or diastolic > 90 mmHg) [4,5].

The 2005 National Cholesterol Education Program (NCEP) Adult Treatment Panel III (ATP) criteria for diagnosing MetS require the presence of at least three of the following risk factors: blood glucose levels greater than 5.6 mmol/L (100 mg/dL) or medication for elevated glucose; HDL cholesterol levels below 1.0 mmol/L (40 mg/dL) in men or 1.3 mmol/L (50 mg/dL) in women, or medication for low HDL-C; triglyceride levels above 1.7 mmol/L (150 mg/dL) or medication for high triglycerides; a waist circumference greater than 102 cm in men or 88 cm in women; and blood pressure higher than 130/85 mmHg or medication for hypertension [4].

American Heart Association/National Heart, Lung, and Blood Institute (AHA/NHLBI) published a unified definition of MetS that incorporated a variety of factors. The IDF established MetS using waist circumference as a primary criteria, requiring a waist measurement more than 94 cm in males and 80 cm in women. In addition, the presence of two or more of the following was needed: blood glucose levels above 5.6 mmol/L (100 mg/dL) or a diagnosis of diabetes; HDL cholesterol levels below 1.0 mmol/L (40 mg/dL) in men or 1.3 mmol/L (50 mg/dL) in women, or medication for low HDL-C; triglyceride levels above 1.7 mmol/L (150 mg/dL) or medication for high triglycerides; and blood pressure higher than 130/85 mmHg or medication for hypertension. This definition is similar to the NCEP ATP III criteria, with the main difference being the waist circumference thresholds [5].

In 2003, many organizations, notably the European Group for the Study of Insulin Resistance (EGIR) and the American Association of Clinical Endocrinologists (AACE), established different definitions of MetS. The variety of these criteria and their associated cut-off values has resulted in substantial variation in epidemiological data on MetS. This variation emphasizes the crucial need for uniform criteria and procedures to improve the precision of epidemiological evaluations and broaden our understanding of MetS across varied communities.

MetS and Liver Involvement

Initially, Reaven did not include obesity in his description of Syndrome X since he found that that IR could occur in non-obese people and that some obese people were insulin sensitive. Obesity evaluated only by Body Mass Index (BMI) is not a valid predictor of MetS without taking into account other characteristics such as waist circumference (WC), age, gender, and ethnicity. This is because IR has a stronger association with visceral adipose tissue than with total body fat. Visceral adipose tissue, which is found in deep abdominal compartments, is more metabolically active than subcutaneous fat in the surface layers. Adipocytes (fat cells) in these deep compartments produce more free fatty acids and inflammatory cytokines, both of which are associated with insulin resistance and other MetS components. As a result, assessing fat distribution, particularly the amount of visceral fat, is critical for appropriately evaluating the risk of MetS and associated metabolic abnormalities. In this respect, computed tomography (CT) investigations have revealed that an excessive buildup of visceral adipose tissue is a major predictor of IR [6]. However, because to radiation concerns and the expensive cost of CT scans, this technology is unlikely to be extensively employed for adiposity measurement. Recent study has revealed that abnormalities linked with visceral obesity are nearly comparable to those seen in those with excess liver fat [7]. Non-invasive methods, such as magnetic resonance spectroscopy, have been developed to accurately assess hepatic fat buildup, which correlates well with liver biopsy results. This is presently the best non-invasive approach for measuring hepatic triglyceride content (HTGC) and detecting hepatic steatosis. In contrast to subcutaneous adipose tissue, liver fat is strongly associated with fasting insulin levels and direct assessments of hepatic insulin sensitivity. A fatty liver promotes the overproduction of glucose and lipids, notably very low-density lipoproteins (VLDL), which are important components of MetS. It also causes a rise in certain cardiovascular risk factors, such as fibrinogen, CRP, PAI-1, and coagulation factors [8].

NAFLD in the Pathogenesis of Metabolic Syndrome

NAFLD is indicated by the accumulation of fat in the liver, with more than 5-10% of hepatocytes being fatty. This condition is not linked to other known causes of liver fat accumulation, such as excessive alcohol consumption (defined as more than 20 grams per day for women and more than 30 grams per day for men according to European and American guidelines), viral infections, medications, toxins, autoimmune disorders, or iron overload. NAFLD is commonly associated with IR. NAFLD ranges from simple fatty infiltration, known as NAFL, which is free of inflammation, to a more severe form, NASH, which includes both fat and inflammation [9]. Over time, NASH can proceed to cirrhosis [10], which can lead to end-stage liver disease (ESLD) or HCC. It is crucial to highlight that not all people with MetS develop NAFLD, and not everyone with NAFLD progresses to NASH.

NAFLD and MetS may be connected in a vicious cycle, with NAFLD functioning as both a symptom and a risk factor for MetS. Histologically, alcoholic steatohepatitis and NASH can seem similar because they both include macrovesicular steatosis with a combination of big and tiny lipid droplets. Both disorders may show expanding necrosis, mild inflammation, and fibrosis. The gold standard for diagnosing these disorders is still liver biopsy, which allows for the detection of certain histological characteristics.

Currently, it is believed that around 25% of the worldwide population has NAFLD. Approximately 25% of people with NAFLD are considered to have developed NASH [11]. Epidemiological studies reveal that around 82% of NASH patients are obese, 83% have hyperlipidemia, and 48% have type 2 diabetes [11]. Longitudinal follow-up studies show that teens diagnosed with NAFLD/NASH have a higher risk of cirrhosis and death than the general population of the same age [12].

Diagnosis and Detection Methods of NASH

NASH itself can often be asymptomatic, although patients with a high body mass index (>25 kg/m²) and T2DM features such as hyperglycemia and insulin resistance are encouraged to be screened for the presence of fatty liver disease [13].

Nevertheless, a recent population study has highlighted that NASH patients have a higher incidence of fatigue and abdominal discomfort, which are shown to be correlated with hepatic lobular inflammation [14]. This may be because hepatic inflammation is associated with elevated plasma inflammatory cytokines [15], which creates a metabolically inflamed milieu that can negatively affect the mood [14].

Patients consuming less than the excessive alcohol intake threshold of >20–30 g/day are classified as having NAFLD, and patients who consumed above that threshold would be diagnosed as having alcoholic fatty liver disease (AFLD), typically treated by alcohol abstinence [16]. Although NAFLD/NASH is not a result of excessive alcohol intake, it shares many histological similarities with AFLD, such as liver steatosis and inflammation [17]. Nevertheless, it might not be possible to determine whether low alcohol use contributes to the development of NAFLD/NASH [16].

Elevation in the plasma of the liver enzymes alanine transaminase (ALT) and aspartate aminotransferase (AST) in a routine blood test is generally the first line of diagnosis [18]. Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) are predominantly expressed in hepatocytes. During hepatocyte necrosis, these enzymes leak into the bloodstream, serving as biomarkers for liver injury. However, patients with NASH may present with normal plasma levels of ALT and AST. Additionally, other conditions, such as viral hepatitis, can also lead to elevated levels of these enzymes, complicating the interpretation of liver function tests. ALT and AST are thus insufficiently specific and sensitive enough to determine the presence or severity of NASH [19]. To confirm the presence of fatty liver, computed tomography (CT) scan or MRI can potentially be used as a non-invasive diagnostic tool to assess the percentage of fat in the liver [19]. However, using MRI as a diagnostic tool in the clinic may not be practical due to the high cost and limited availability. Patients in rural/regional areas and/or from low socioeconomic areas would be unlikely to be able to access it [19]. Importantly, the percentage of hepatic fat alone does not accurately reflect the degree of liver inflammation, hepatocyte injury, or tissue fibrosis. Consequently, histological scoring of liver biopsy specimens by pathologists remains the gold standard for assessing the presence and severity of NASH. In this evaluation, liver tissues are stained with hematoxylin and eosin and assessed using an ordinal scoring system: steatosis is scored from 0 to 3, inflammation from 0 to 3, and ballooning degeneration of hepatocytes from 0 to 2. The ordinal scores for these three parameters are then combined to generate a total NAFLD Activity Score (NAS), which provides a comprehensive measure of disease activity. In both clinical and preclinical studies, there is a general consensus that a total NAS ≥5 is classified as definitive NASH rather than a simple fatty liver disease [20]. Despite its status as the gold standard for diagnosing NASH, the invasive nature of liver biopsy has rendered this histological diagnostic approach less favorable among clinicians and patients. Consequently, there is a pressing need for further research aimed at elucidating the molecular mechanisms underlying NASH. Such investigations could facilitate the identification of sensitive and highly specific biomarkers for NASH, potentially enabling non-invasive diagnostic alternatives. By advancing our understanding of the pathophysiological processes involved in NASH, these studies may ultimately contribute to improved disease stratification, monitoring, and therapeutic interventions.

Prognosis in MetS

MetS patients' prognosis is impacted by the severity of their separate components as well as the occurrence of cardiovascular problems. Cardiovascular problems predict a poor prognosis in MetS patients, and the risk of cardiovascular complications is thought to be exacerbated by the combination of various ASCVD risk factors [21]. Patients with MetS have a more than twice increased likelihood of experiencing cardiovascular events than those without the condition. This elevated risk is mostly caused by MetS' underlying components, including dyslipidemia, hypertension, and insulin resistance, all of which lead to the development of atherosclerosis and other cardiovascular issues. As a result, this increased risk is a strong predictor of a poor long-term prognosis, underscoring the importance of early intervention and treatment techniques to reduce cardiovascular morbidity and death in this group. However, recent advances in treating atherosclerotic cardiovascular diseases have resulted in significant improvements in outcomes [22].

Non-Invasive Assessment of NASH

An ideal biomarker should be objectively quantifiable, represent essential disease processes, and correspond to illness progression. Biomarkers in NAFLD aid in diagnosing steatosis, assessing for NASH, and quantifying fibrosis. Identifying patients with symptoms that contribute to liver disease development is critical owing to the danger of cirrhosis and accompanying consequences. While liver biopsy is the gold standard for diagnosing and staging NASH, it is invasive, expensive, and prone to variation. To diagnose NASH and stage liver fibrosis, doctors are developing noninvasive tools such as prediction models, blood biomarkers, and imaging-based procedures.

7.1. Clinical and Laboratory Variables (Serum Biomarkers)

In a large-scale study of 125,052 hospitalized patients with NAFLD or NASH in France, around 10% had compensated or decompensated cirrhosis upon diagnosis [23]. Notably, NASH has been related to fast disease progression, with 27.5% of initially compensated patients progressing to decompensated cirrhosis within 7 years. These findings emphasize the urgent need for robust, less invasive serum biomarkers to identify fibrosis early in the course of NASH. The performance of such non-invasive biomarkers should be rigorously evaluated and compared to liver histology to ensure accurate fibrosis assessment. Plasma cytokeratin 18 (CK18) fragments, which indicate hepatocyte apoptosis, have been extensively studied as a marker for steatohepatitis, albeit with modest accuracy [24]. Recent research has identified a positive association between soluble CD163, a macrophage activation marker, and histologically confirmed NASH fibrosis in two independent NAFLD patient cohorts from Australia and Italy. This suggests that sCD163 may hold potential as a biomarker for NASH fibrosis [25]. Over the last 15 years, new diagnostic panels have been established to increase illness diagnosis accuracy, recognizing that individual indicators such as cytokeratin 18, inflammatory markers (e.g., TNF, IL-8), and hormones (e.g., adiponectin, FGF21) provide only minimal accuracy on their own. Among them, Pelekar et al.,[26] developed a diagnostic panel with six variables: age, gender, AST, BMI, AST/ALT ratio, and serum Hyaluronic Acid. This model has an area under the receiver operating characteristic curve (AUROC) of up to 0.76, suggesting a moderate degree of diagnostic accuracy. The NASH Test is a proprietary diagnostic tool that uses 13 clinical and biochemical variables—including age, gender, height, weight, and serum markers such as triglycerides, cholesterol, and liver enzymes—to determine the presence of NASH. It has high specificity (94%) and a moderate AUROC of 0.79, but lower sensitivity (33%), a positive predictive value of 66%, and a negative predictive value of 81% [27-29]. However, the test's practical application is limited because it requires several variables that are not commonly measured in routine clinical setsettings. Fibrosis-4 index (Fib-4), initially developed for patients co-infected with HCV and HIV, has been validated for use in those with NAFLD by Shah et al.,[30]. The Fib-4 score relies on commonly available variables: age, ALT, AST, and platelet count. It has demonstrated an area under the receiver operating characteristic curve (AUROC) of 0.80 for identifying advanced fibrosis. The test has positive and negative predictive values of 80% and 90%, respectively, for Fib-4 scores of 2.67 and 1.30. Additionally, Fib-4 is effective in accurately excluding patients with F2 fibrosis. Younoussi [31] created the NASH Diagnostic Panel, which seeks to predict NASH using a model that combines characteristics such as diabetes, gender, BMI, triglycerides, and particular cytokeratin markers such as M30 (indicating apoptosis) and M65-M30 (indicating necrosis). For predicting NASH-related fibrosis, the panel incorporates these predictors and achieachievesrea under the curve (AUC) of 0.80 with a 95% confidence interval of 0.68–0.88 and a p-value of <0.00014. For advanced fibrosis specifically related to NASH, the model includes type 2 diabetes, serum triglycerides, tissue inhibitor of metalloproteinases-1, and AST, with an AUC of 0.81, a 95% confidence interval of 0.70–0.89, and a p-value of 0.0000.000062.ent validated serum test for differentiating mild to moderate fibrosis (F0–F2) from advanced fibrosis (F3–F4) in NAFLD patients measures concentrations of alpha2-macroglobulin (A2M), hyaluronic acid (HA), and tissue inhibitor of metalloproteinases-1 (TIMP-1). This test demonstrated an area under the receiver operating characteristic curve (AUROC) of 0.85, with a 95% confidence interval of 0.82–0.89. At a predetermined cutoff score of 17, it achieved a sensitivity of 79.7% (95% CI, 71.9–86.2%) and specifa specificity.7% (95% CI, 71.8–79.4%). The test performed particularly well in diagnosing cirrhosis, with negative predictive values ranging from 92.5% to 94.7% across validation cohorts. It correctly classified 90.0% of F0 samples, 75.0% of F1 samples, 77.4% of F3 samples, and 94.4% of F4 samples [32,32]. He past 15 years, numerous minimally invasive blood tests for liver fibrosis have been introduced. However, it's important to recognize that these serum biomarkers tend to be more effective in detecting advanced stages of liver fibrosis rather than early stages. Among the most promising biomarkers currently under evaluation in clinical trials for emerging treatments are alpha2-macroglobulin (A2M), hyaluronic acid (HA), tissue inhibitor of metalloproteinases-1 (TIMP-1), cytokeratin 18 (CK18), and the Fibrosis-4 (Fib-4) index. These biomarkers are crucial for advancing non-invasive diagnostic methods and monitoring liver fibrosis as new therapies develop.

7.2. Imaging-Based Biomarkers

Transient elastography (TE) assesses liver stiffness by measuring the velocity of an elastic shear wave produced by pulse-echo ultrasound [33]. The velocity of this wave, indicated in meters per second, correlates with tissue stiffness, which in turn shows the degree of fibrosis—the stiffer the tissue, the quicker the wave propagates. While TE is useful for measuring hepatic stiffness, it does have limits. It may be unreliable in patients with considerable fat or fluid between the chest wall and the liver, as well as in obese people, with a failure rate of around 20%. High transaminase levels, sinusoidal congestion, and extrahepatic cholestasis can all have an impact on TE outcomes, and the approach is prone to fluctuation [34]. A measurement is considered valid if it yields at least ten valid readings and the interquartile range is less than 30% of the median liver stiffness [35]. Results are presented in kilopascals (kPa), with typical values around 5 kPa. The AUROC values for diagnosing advanced fibrosis are 0.88 for the FibroScan M probe and 0.85 for the XL probe, with the XL probe designed for obese patients showing comparable accuracy to the M probe.

Point Shear Wave Elastography (pSWE), also known as Acoustic Radiation Force Impulse (ARFI) imaging, is a sophisticated ultrasound method that quantifies liver stiffness by creating shear waves using short-duration acoustic pulses. This approach uses the propagation of these shear waves inside the hepatic tissue to produce a non-invasive assessment of tissue elasticity, which is associated with liver fibrosis and overall hepatic health. These pulses cause localized micrometer-scale displacements in liver tissue [36], which are subsequently measured to determine stiffness. While pSWE has been proven to be as effective as TE in identifying severe fibrosis, particularly F4 stages, it has not been widely researched for diagnosing NASH. Further large-scale study is required to address concerns such as the restricted measurement range and differences between results from various liver lobes, which can have an impact on diagnostic accuracy.

Magnetic Resonance Elastography (MRE) assesses liver stiffness by analyzing mechanical vibrations sent through liver tissue, comparable to ultrasound-based methods. MRE uses a modified phase-contrast approach to image the propagation of shear waves in the liver, which is unaffected by body habitus [37]. It is regarded as the most accurate test for detecting liver fibrosis. However, its use is limited due to factors such as high costs, examination length, and availability. A meta-analysis of nine trials, including 232 NAFLD patients, indicated that MRE can correctly diagnose fibrosis at all phases, with AUROC values ranging from 0.86 to 0.91, regardless of liver inflammation or body mass index (BMI) [38].

LiverMultiScan (Perspectum Diagnostics, Oxford, UK) is a recent multiparametric MRI technology that provides complete imaging of liver diseases. This methodology incorporates three methods: T1 mapping to assess fibrosis and inflammation, T2 mapping to quantify liver iron, and magnetic resonance spectroscopy (H-MRS) to measure liver fat [39]. A key innovation in this method has been the invention of iron-corrected T1 mapping, which tackles the issue of "pseudo-normal" T1 results generated by high iron levels in the context of fibrosis. This innovation increases the technique's application and accuracy [40]. However, more validation studies are required to prove its efficacy and dependability in clinical practice.

7.3 Emerging Circulating Biomarkers

Circulating extracellular vesicles (exosomes and ectosomes) contain various cellular molecules such as proteins, mRNA, miRNAs, and DNA and can serve as biomarkers in NAFLD and NASH [41]. Following their release into the intercellular space, ectosomes bind to recipient cells and deliver their informative cargo [41]. The recipient cells may then undergo epigenetic reprogramming and subsequent phenotypic alterations according to the molecular information received [41]. In patients with NAFLD, Kornek et al. employed fluorescence-activated cell sorting (FACS) and observed an increase in ectosomes associated with surface markers from monocytes and natural killer cells, alongside a reduction in neutrophils and leuco-endothelial cells. Production of exosomes and other extracellular vesicles are increased in patients with NASH [42]. Recently, it has been suggested that a specific protein signature in blood extracellular vesicles may be used to diagnose NASH noninvasively [43]. It has been hypothesized that exosomes play a crucial role in the communication between hepatocytes and hepatic stellate cells. In the context of lipotoxic injury, hepatocytes damaged by fatty acids produce exosome-like vesicles that are subsequently taken up by hepatic stellate cells, promoting their fibrogenic activation. In transgenic mice, increased levels of CD10 protein in urinary exosomes was associated with steatosis and fibrosis [44].

In addition to proteins, other biomarkers associated with extracellular vesicles, such as circulating nucleic acids, have been identified, including DNA fragments commonly referred to as cell-free DNA (cfDNA) and RNA. Current research on cfDNA emphasizes specific characteristics rather than quantification, with DNA methylation being a key area of interest. Hardy et al. showed an increase in cfDNA methylation at the PPARgamma gene promoter in a cohort of patients with NAFLD as compared to healthy controls [45]. This is an important requirement for HSC activation and appears correlated with fibrosis progression in NAFLD. However, the preliminary results indicate substantial lack of specificity as this correlation has been observed also in HBV chronic infection [45].

Cell free noncoding RNA comprises long (lncRNA) and short (miRNA) species of RNA. The lncRNAs are cell-specific molecules with a length of >200 bp and with several of the theorical essential characteristics of good biomarkers, including accessibility, although it is difficult to process them and to obtain accurate measurements [46,47]. Recent in vitro studies reported aberrant expression of lncRNA during HSC activation, and this was confirmed in two in vivo studies examining liver tissue of NAFLD and NASH patients [48-50]. In biopsy-proven NAFLD patients with disease of different severity, another small study showed difference in the expression profile of patients when compared to normal liver [51].

The miRNAs, utilized for intercellular signal transduction, are small noncoding microRNAs with epigenetic functions able to transcriptionally regulate gene expression [52]. MiRNAs contribute to the pathogenesis of NAFLD/NASH at various levels of disease development and progression and probably are the most extensively studied epigenetic modifications in NAFLD. In particular, miR-122 has a well-established role in lipid metabolism and was shown upregulated in NASH in comparison to NAFLD [52]. In NASH, the exosome-packaged liver specific miR-122 increases over time and correlates with histological severity. Some studies have demonstrated that, in the liver of patients with NASH, miR-122 is downregulated [52]. Using immunoprecipitation, it was shown that, in NASH, the low intrahepatic levels of miR-122, rather than a real downregulation, may be a consequence of the increased rate of release into the circulation [53]. It can be postulated that exosomes derived from hepatocytes facilitate the sensitization of macrophages to inflammatory signals through the action of microRNA-122 (miR-122). Additionally, concentrations of other microRNAs, such as miR-34a, have been found to be significantly elevated in patients with non-alcoholic fatty liver disease (NAFLD). Recent investigations have identified a specific microRNA signature capable of distinguishing between alcoholic steatohepatitis (ASH), NAFLD, NASH, and cholestatic liver disease.

While these approaches show promise, their clinical implementation faces challenges. Studies focusing on cell-free DNA (cfDNA) are constrained by the very low concentrations and high fragmentation of cfDNA, whereas investigations into long non-coding RNAs (lncRNAs) are limited by small sample sizes and a lack of standardized protocols.

Microbiota-Related Biomarkers

Profiling gut microbiota and its metabolites has emerged as a viable non-invasive technique for the early detection of liver illnesses such as NAFLD, NASH, and hepatic fibrosis. This technique takes use of the intricate interaction between the gut microbiota and liver function, providing insights into disease pathophysiology and possible indicators for early identification.

According to research, dysbiosis (also called dysbacteriosis), defined as a drop in Firmicutes, an increase in Proteobacteria, and a decrease in Ruminococcus at both genus and species levels, is connected with the prevalence of NASH and fibrosis in people. Various microbial and metabolic alterations may act as biomarkers for the early diagnosis and monitoring of various liver diseases [55].

In a study of 86 individuals with biopsy-confirmed NAFLD, researchers examined both stool microbiomes and serum metabolomes to discover 37 species associated with progressive fibrosis [56]. This resulted in the development of a prediction algorithm with great accuracy (AUROC 0.936) for diagnosing advanced fibrosis. Further analysis of serum metabolites influenced by gut bacteria revealed significant differences in 11 amino acids and metabolites involved in nucleoside and carbon metabolism. These findings imply that future serum tests based on gut microbiome profiles may provide useful markers for diagnosing advanced fibrosis.

Conclusions

NAFLD and its development to NASH are major problems in the setting of MetS, affecting global health with growing prevalence and complicated pathophysiology. The NAFLD spectrum, which ranges from simple steatosis (NAFL) to advanced NASH, highlights the various degrees of liver injury and inflammation that can lead to severe outcomes such as cirrhosis and hepatocellular carcinoma. The interaction between NAFLD and MetS, defined by insulin resistance and metabolic dysregulation, reveals a cyclical connection where each illness exacerbates the other, complicating therapy and prognosis.

NASH is difficult to diagnose since it is asymptomatic and existing diagnostic approaches have limitations. While liver biopsy is conclusive, it is invasive and is not widely used in clinical practice. Non-invasive technologies, such as imaging techniques and serum biomarkers, are potential alternatives, but they have limits in sensitivity, specificity, and accessibility. Techniques such as TE and MRE have demonstrated great diagnostic accuracy, but they are restricted by cost and practicality, especially in resource-constrained environments.

Emerging non-invasive diagnostics, including serum biomarkers, extracellular vesicles, and microbial profiles, have the potential to diagnose NASH sooner and with greater precision. Biomarkers such as cytokeratin 18 fragments, soluble CD163, and advanced fibrosis panels have different degrees of accuracy, indicating the need for further refining and validation. Furthermore, the combination of gut microbiota profiling and circulating nucleic acids, such as miRNAs and cfDNA, represents a new frontier in NASH diagnostics, though these methods still require validation and standardization.

Overall, while advances in diagnostic tools and biomarkers are encouraging, they highlight the need for ongoing research to produce reliable, cost-effective, and easily accessible techniques for detecting and treating NASH. Early identification and treatments remain critical to avoiding progression and improving patient outcomes in the rising population afflicted by MetS and liver disorders.

Declarations

Author Contributions

All authors have contributed equally.

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Competing interests

The authors declare that they have no competing interests.