2.1. Relation between Hepatic Steatosis and Insulin Resistance

The association between NAFLD and IR has been widely investigated. The prevalence of IR is high in NAFLD and even higher in subjects with NASH compared to those with simple steatosis ^[2]; to date, IR is considered the main pathogenetic mechanism involved in the onset of NAFLD and its progression to NASH ^[3][4][5]. In the IR state, fat accumulation in the liver is caused by the impaired uptake, synthesis, export and oxidation of FFAs. In NAFLD subjects, the amount of hepatic steatosis correlates with increased plasma levels of FFAs due to the impaired suppression of lipolysis from the adipose tissue; subcutaneous adipose tissue represents a major source of intrahepatic fat (~62–82% of intrahepatic triglycerides). This mechanism is independent of obesity and diabetes, as it has also been demonstrated in nonobese, nondiabetic NAFLD patients; in the latter group, IR affects HGP, glucose disposal (glycogen synthesis and glucose oxidation), lipolysis and lipid oxidation. Although visceral fat is not the main supplier of circulating FFAs, it represents the main source of inflammatory cytokines reaching the liver, as confirmed by the correlation between IL-6 and C-reactive protein levels in the portal vein ^[6][7].

In the insulin-resistant condition, the liver loses its ability to suppress HGP in response to insulin and enhances DNL through the activation of the Notch signaling pathway ^[8]. This explains, on one hand, the increase in DNL that, in NAFLD patients, is 5-fold higher when compared to that in healthy subjects (26 vs. 5%, respectively) and, on the other hand, the predisposition to diabetes in subjects with NAFLD ^[8].

2.2. Insulin Resistance in the Progression from Simple Steatosis to Nonalcoholic Steatohepatitis and Fibrosis

The development of NASH has been linked to a variety of factors, including nutrient intake, endocrine derangements (insulin, leptin, adiponectin and ghrelin), alterations in gut microbiota (endotoxemia) and epigenetic factors, possibly acting on a genetic predisposition ^[9]. Unfortunately, the molecular mechanisms leading to NASH and fibrosis development have not been fully elucidated yet. Hepatic triglyceride depositions into lipid droplets are considered a sort of inert storage; notwithstanding this, excessive lipid overload may enhance lipid oxidation and reactive oxygen species (ROS) release, making the liver susceptible to the action of proinflammatory factors. In obese and in diabetic patients, ROS levels correlate with the C-reactive protein concentration as well as fibrinogen levels, suggesting a subclinical proinflammatory state ^[9][10]. In NAFLD, the saturated FFA palmitate seems to play an important role in the progression of liver damage; it is synthesized in the DNL pathway and is able to trigger fibrogenesis in the liver through the activation of hepatic macrophages ^[11].

Several factors are able to mediate liver damage in patients with NAFLD; some of these are synthesized by the liver, while others are released by the adipose tissue and exert their effects in a paracrine way ^[12]. The liver is the main source of selenoprotein P (SeP), a selenium carrier protein with antioxidant properties. SeP is regulated by hyperglycemia and is able to induce IR, disrupting glucose homeostasis, thus favoring the development of T2DM ^[12]. Recently, we found that circulating SeP increases according to the degree of hepatic steatosis and to the stage of fibrosis in nondiabetic patients with NAFLD, suggesting its potential role in the onset of NASH and progression to fibrosis ^[13].

Leptin is a peptide hormone that is released by adipocytes and plays a role in the regulation of food intake and bodyweight. In the setting of NAFLD, leptin may be expressed by activated hepatic stellate cells (HSCs) and by Kupffer cells (KCs), contributing to hepatic fibrogenesis, thus enhancing HSC signal transduction ^[9][14]. Specifically, leptin stimulates the transcriptional activation of both the α1(I) and α2(I) fibrils, which are major components of dense extracellular matrix (ECM). Furthermore, leptin promotes the synthesis of the matrix metalloproteinase-2 (MMP-2), tissue inhibitor matrix metalloproteinase 1 (TIMP-1), TIMP-2, and alpha-smooth muscle actin (α-SMA) transcripts, all involved in the pathogenesis of liver fibrosis ^[15][16]. Finally, leptin protects HSCs against apoptosis ^[15]. Although higher leptin levels were found in patients with NAFLD compared to healthy controls, its role in the pathogenesis of NASH has not been fully elucidated.

Insulin-like growth factor 1 (IGF-1) is a hormone very similar to insulin in its molecular structure. IGF-1 is expressed primarily by the liver under the control of growth hormone, and it circulates linked to IGF-binding protein 3 (IGFBP-3). IGF-1 is involved in hepatocyte differentiation, proliferation and apoptosis ^[17]. A recent meta-analysis showed that IGF-1 levels are reduced in NAFLD patients compared to healthy controls, suggesting a potential role as a therapeutic target ^[18]. Moreover, Hagstrom et al. found low IGF-1 levels in patients with severe fibrosis (F ≥ 3) compared to those with absent/mild fibrosis ^[19]. Even though the molecular mechanisms linking IGF-1 and the progression of liver damage in the setting of NAFLD have not been elucidated yet, recent data describe a novel role of IGF-1 in regulating stress-induced hepatocyte premature senescence in liver fibrosis. Specifically, IGF-1 is able to attenuate the oxidative stress-induced premature senescence of hepatocytes in mice through the inhibition of the interaction between nuclear p53 and progerin, a truncated version of the lamin A protein, improving hepatic steatosis and fibrogenesis ^[20].

The liver is the main target for adiponectin, the most abundant adipocytokine synthesized by the adipose tissue ^[21]. Low adiponectin levels are associated with steatosis, inflammation and fibrosis in the liver ^[21]. Specifically, circulating adiponectin decreases in obese subjects as well as in fibrotic patients with NAFLD. Adiponectin exerts an antifibrotic action by reducing HSC activation and proliferation; in addition, it favors matrix degradation, reducing the molecular ratio of MMP-1 to TIMP-1, antagonizing leptin-mediated signaling in hepatic fibrogenesis ^[22]. This potent profibrogenic effect of leptin may contribute to the endothelial alteration of hepatic sinusoids, whose fenestrations are progressively replaced by an organized basement membrane. This process, known as the “capillarization” of hepatic sinusoids, is one of the major parenchymal alterations that drive the liver to an architecture causing portal hypertension ^[23][24].

In the past few years, genome-wide association studies have led to the identification of several genes related to NAFLD, NASH, and their complications including hepatocellular carcinoma (HCC). In 2008, Romeo et al. ^[25] described a single-nucleotide polymorphism (SNP) in the patatin-like phospholipase domain-containing 3 (PNPLA3) gene, which encodes the triglyceride lipase adiponutrin and strongly affects fat accumulation in the liver through mechanisms independent of IR. The PNPLA3 rs738409 (G) risk allele, found in ~40% of the European population, can also increase, threefold, the risk of progression to NASH and, most importantly, twelve-fold, the risk of developing HCC ^[26].

In subsequent years, other SNPs have been associated with increased hepatic fat accumulation and, thus, the progression of liver disease. The most important genetic variants are the rs58542926 C > T located in the transmembrane 6 superfamily member 2 (TM6SF2) gene and the rs641738 C > T located in the membrane-bound O-acyl-transferase domain-containing 7 (MBOAT7) gene, which favor hepatic fat accumulation in intracellular lipid droplets via different mechanisms, increasing the susceptibility to inflammation, NASH and fibrosis ^[27].