Nonalcoholic fatty liver disease (NAFLD) is the name given to a spectrum of liver disorders, histologically categorized as simple (i.e., isolated) steatosis or nonalcoholic steatohepatitis (NASH) with or without fibrosis, often resulting in cirrhosis and hepatocellular carcinoma (HCC) [1]. Like its major risk factors, obesity and type 2 diabetes mellitus (T2DM), NAFLD results from the interaction between environmental and genetic factors [2]. Specifically, variants in two genes coding for proteins involved in lipolysis and lipogenesis, namely adiponutrin (also known as patatin like phospholipase domain containing 3 [PNPLA3]) and, more recently, 17β-hydroxysteroid-dehydrogenase type 13 (HSD17B13), have been shown to play a significant role in NAFLD pathogenesis and progression [3].

There is an ongoing debate regarding whether screening campaigns should be conducted to identify NAFLD individuals [4]. The overwhelmingly high prevalence, the relatively low risk of progression to significant liver disease for most NAFLD patients, and the paucity of treatment options other than lifestyle measures—notoriously difficult to adhere to—make large-scale screenings for NAFLD highly unsuitable for the general population. Thus, screening efforts focused on individuals at higher risk of liver disease progression are likely to be far more rewarding. In this regard, T2DM patients are known not only to be at high risk for NAFLD development [5] but also to display a progression rate toward significant liver fibrosis double that observed in other NAFLD patients [6]. However, it is still unclear what are the determinants of this increased risk given the multifactorial etiology of diabetes [7]. Certainly, the perception of NAFLD among physicians caring for patients with T2DM varies widely, with normal aminotransferase test being a predictor for non-referral or no further diagnostic exploration of T2DM patients among endocrinologists [8].

In recent years, the way clinicians approach a patient with suspected liver disease has been changed by the advent of transient elastography (TE), a non-invasive technique which—by extrapolating the likelihood of fibrosis from liver stiffness (LS)—has greatly reduced the indications for a liver biopsy, perceived by many as unduly aggressive and costly [9]. Conveniently, new TE machines have been implemented with a software able to quantify liver fat accurately, by measuring the controlled attenuation parameter (CAP). CAP identifies steatosis independently of the presence of fibrosis, which is not the case for ultrasound (US) [10]. Systematically performing TE with CAP measurement on patients with T2DM may thus offer the opportunity of unveiling the true burden of liver disease in this condition, thereby increasing the awareness of its existence among non-hepatologists. At the same time, it makes possible exploring genetic and environmental risk factors associated with NAFLD progression towards liver fibrosis among diabetics. With these aims in mind we performed the present study.

In the present study, we show that—in an unselected cohort of non-morbidly obese T2DM patients—the prevalence of severe NAFLD is substantial. Anthropometric characteristics of patients explain most of the variability observed, with a significant contribution afforded by genes controlling lipogenesis and lipolysis. Remarkably, age and duration of T2DM appear not to contribute significantly to the prevalence of NAFLD. These findings and their possible clinical implications in the context of existing literature are discussed below.

The epidemic diffusion of obesity and T2DM, along with the improvement in HCV eradication thanks to antiviral agents, has made NAFLD the main cause of chronic liver disease and cirrhosis worldwide [19]. According to a recent projection, by 2030 the prevalence of NASH will have increased by 15% to 56%, while liver mortality and significant liver disease will have more than doubled as result of an aging/increasing population [20].

The natural history of NAFLD can vary significantly from patient to patient. Typically, NAFLD is a slowly progressive disease, with the vast majority of NAFLD patients never developing end-stage liver disease. The rate of progression is equal to one stage of fibrosis every 14 years in NAFLD, and every 7 years in NASH. However, in 20% of cases fibrosis progresses more rapidly [21]. Therefore, a significant proportion of NAFLD patients will experience liver-related complications, thus making an accurate risk prediction even more difficult.

In this context, T2DM patients are of major interest. First of all, the prevalence of NAFLD is higher in T2DM patients than in the general population [22]. Moreover, patients with established T2DM are more likely to suffer from more severe histological forms of NAFLD, even in cases where aminotransferase serum levels are normal [23], being aminotransferase levels an unreliable marker of NAFLD among patients with T2DM. Finally, the coexistence of NAFLD and T2DM typically worsens the course of both diseases [24].

However, despite the importance that should be given by clinicians to the diagnosis and treatment of NAFLD in T2DM patients, the usefulness of screening strategies to detect NAFLD has been recently questioned given our incomplete understanding of the natural history of this disease.

In our study, we highlight the essential contribution of excess body weight to liver fibrosis progression. In good agreement, a study on a cohort of 1,527 T2DM and NAFLD patients has rerecently shown that BMI is a predictor of FIB-4 score both at baseline and after 3 years of follow-up [25]. The reason why obesity negatively impacts liver functions is probably due to the fact that the adipose tissue, in obese subjects, acquires a proinflammatory, profibrogenic, and proangiogenic phenotype, resulting in abnormal production of adipokines and cytokines (e.g., leptin, interleukin [IL]-1, IL-6, and tumor necrosis factor-α [TNF-α]) involved in liver pathogenesis. For instance, leptin contributes to neoangiogenesis and directly modulates hepatic stellate cells in a profibrogenic manner [26]. Contextually, chemokines and proinflammatory cytokines, such as IL-1, IL-6, and TNF-α mediate macrophage infiltration, which causes adipose tissue “inflammation” in obese subjects, resulting in insulin resistance and deregulated secretion of adipokines [27]. Interestingly, obesity contributes not only to liver fibrosis progression but also to the complications brought by cirrhosis. In this regard, obesity is an independent risk factor for clinical cirrhosis decompensation, where dysregulated cytokines could contribute to worsening intrahepatic resistance and portal hypertension [28,29]. Moreover, obesity is a well known independent risk factor for the development of HCC [30]. Finally, hypovitaminosis D is highly prevalent in obese subjects due to the fact that vitamin D is a fat-soluble molecule stored in the adipose tissue [31]. In this regard, the severity of NAFLD has been inversely correlated with plasma vitamin D levels [32], possibly because vitamin D plays an antifibrogenic and anti-inflammatory role [33], similar to what observed in other inflammatory diseases [34].

Besides environmental factors and comorbidities, other genetic and epigenetic predictors of liver disease progression have been identified in recent years [35]. The strongest genetic predictor of chronic liver disease progression is the SNP rs738409 in PNLPA3 gene. This gene encodes a membrane bound triacylglycerol lipase that mediates triacylglycerol hydrolysis. As PNPLA3 mutation has been robustly associated to steatosis, fibrosis/cirrhosis, and HCC on a background of metabolic, alcoholic, and viral insults [36], in the present study we sought to determine whether PNPLA3 mutation was associated with severity of liver disease. Previously, an increased risk of liver fibrosis was reported in T2DM patients carrying the mutated variant of rs738409 [37], although the estimation of liver fibrosis was based upon Fibrotest rather than on LS. From our data, we confirm the existence of a significant trend toward increasing LS in heterozygotes or mutated homozygotes over WT individuals. In particular, our results indicate that in T2DM patients PNLPA3 is associated with a significant risk of liver disease progression, although the strength of this association might be less relevant than in the general population probably because mainly driven by excess BMI and, by extension, body fat.

Another controversial point is that the association between PNPLA3 and liver fibrosis does not automatically imply a causal relationship. However, the observation that insulin regulates PNPLA3 gene expression [38] strongly argues in favor of a potential link between liver fibrosis progression and adiponutrin. Fittingly, adenovirus-mediated liver expression of a missense mutant form of PNPLA3 in mice was sufficient to abolish the hydrolase activity of adiponutrin leading to triglyceride accumulation, suggesting a possible cause-effect relationship between PNPLA3 polymorphism and development of steatosis and fibrosis [39]. Furthermore, in humans the missense mutation leads not only to intrahepatic remodeling but also to impairment of the fat efflux pathway from the liver, which seems to be caused by a reduction in very low density lipoproteins secretion [40].

It is also important to consider that PNPLA3 is expressed by human hepatic stellate cells, where adiponutrin catalyzes the hydrolysis of retinyl esters [41]. Consistently, the missense mutation of PNPLA3 gene is associated with lower circulating concentrations of free retinol [42] and higher intrahepatic concentrations of retinol [43], although whether this may contribute to fibrosis has yet to be defined.

Another important aspect further supporting a cause-effect link between PNPLA3 and liver fibrosis is that NAFLD patients carrying the variant rs738409 allele display prominent activation of the hepatic stem/progenitor cell niche, which is associated with a more aggressive histological pattern (i.e., portal fibrogenesis) and increased oxidative stress [44].

Although the association between PNPLA3 gene and liver steatosis/fibrosis is well established, the pathogenetic mechanism underlying this association still remains unclear. In this regard, the detrimental impact of carrying the variant rs-738409 allele has been recently shown to be mitigated by a concomitant mutation in the HSD17B13 gene in the general population [3]. In this regard, one of the main findings of our study is that the rs72613567:TA mutation is associated with a lower risk of liver fibrosis progression also in case of T2DM, as shown for the general population. We find that the association between the variant rs738409 allele and severe NAFLD is fully maintained in WT HSD17B13 carriers. By contrast, in those patients carrying at least one mutated allele, PNPLA3 does not predict LS any longer. This suggests a potential protective role of such mutation, although the cross-sectional design of our study does not allow drawing definitive conclusions. In this regard, the reason why this mutation seems to play a protective role against liver diseases, mitigating the detrimental effect of the variant rs738409 allele, has yet to be determined. Su et al. [45] showed HSD17B13 WT mRNA upregulation in NAFLD patients. Moreover, overexpression of HSD17B13 in the livers of C57BL/6 mice and cultured hepatocyte cell lines enhanced substantially lipogenesis and increased the number and size of lipid droplets [45]. In the same study, upregulation of HSD17B13 was observed in the livers of both diabetic mice and high-fat diet-fed mice, suggesting that HSD17B13 may play a crucial role in the pathogenesis of fatty liver and may also be relevant in diabetes. Abul-Husn et al. [3] demonstrated in a large liver impairment patient cohort the important effect of HSD17B13 variant. The insertion of “A” in a splice donor site led to a protein-truncating isoform [3]. The same group, through RNA sequencing-based expression analysis demonstrated that HSD17B13 variant was associated with decreased PNPLA3 mRNA expression in an allele-dependent manner. Similarly, the 434K allele of PNPLA3 could mitigate the effect of the PNPLA3 I148M allele on chronic liver disease by reducing hepatic PNPLA3 mRNA and protein expression [46].

As expected, we found a strong correlation between steatosis development and T2DM, implying that the genetic component only exerts a marginal effect. This supports the hypothesis that, while fatty liver is a very common condition in T2DM patients, the development of liver fibrosis is significantly influenced by PNPLA3.

From a clinical standpoint, being obvious that major efforts should be devoted to correct excess body weight in T2DM, we provide further reasons to diabetologists to do so, by adding to the potential benefits of losing weight the reduced risk of progressive liver disease. Furthermore, our findings indicate that PNLPA3 and HSD13B17 products might represent potential therapeutic targets in the prevention of liver disease progression in T2DM patients. Though both variants might be worth of consideration in the construction of prediction models aimed to improve NAFLD detection in patients with T2DM, the inclusion of periodic TE examinations during patient evaluation would result in a cheaper and more direct screening protocol.

Among the several limitations of our study, we must acknowledge the fact that TE is not the ideal method to characterize NAFLD. It is inferior to magnetic resonance based-techniques [15] and has not stood the test of time like liver biopsy. However, it is far more applicable than both techniques to clinical practice. Moreover, being a cross-sectional study we cannot provide date on the effect, if any, that prolonged inadequate control of T2DM might have on fibrosis progression, although punctual data at the time of our study appear not to point in that direction.

In conclusion, patients with T2DM have a high prevalence of liver disease characterized by significant fibrosis, which goes largely undetected in the absence of symptoms and striking laboratory abnormalities. Obesity and, to a minor but not negligible extent, genetic factors contribute to its development.