Hepatic lipid uptake is a function of substrate delivery and transport into the hepatocyte. The major source of FAs for the liver is the systemic plasma FAs pool, mainly derived from the lipolysis of subcutaneous adipose tissue triglycerides, and, to a small extent, from lipolysis of triglycerides in circulating lipoproteins [38,39]. In contrast, only about 5% and 20% of portal vein FAs originate from visceral fat lipolysis in lean and obese subjects, respectively [40]. It has been shown that the rate at which FAs are released into the systemic circulation increases with increasing fat mass in both men and women [41].

Regional mobilization of circulating triglycerides has been demonstrated to be altered in obese patients with NAFLD. Lipoprotein lipase (LpL) hydrolyzes circulating triglycerides, followed by tissue uptake through FA transport proteins. LpL activity appears to be blunted, in response to insulin, in adipose tissue of obese patients. In contrast, NAFLD is associated with increased LpL as well as FA transport proteins [38,39]. Taken together, these data suggest that alterations in adipose tissue lipolytic activity, regional hepatic lipolysis of circulating triglycerides and FA transport proteins are involved in the pathogenesis of hepatic steatosis and ectopic fat accumulation.

The contribution of hepatic de novo lipogenesis in healthy individuals is minor. In fact, in healthy subjects, hepatic de novo lipogenesis in the fasting and postprandial states has been reported to account for less than 5% and 10% of FAs incorporated into intra-hepatic triglycerides (IHTG) and VLDL-triglyceride, respectively [42]. In patients with NAFLD, the rate of hepatic de novo lipogenesis in the fasting state is also quite small and therefore is unlikely to be primarily responsible for the excessive liver fat accumulation. However, in these patients the rate increases in the postprandial state [42,43]. In particular, large increases in de novo lipogenesis following meal ingestion have been shown to precede excessive liver fat accumulation [44]. Importantly, specific dietary habits such as high carbohydrate meal and increased consumption of fructose, have been implicated in this increased rate and therefore in the development of NAFLD and its progression to NASH.

The liver derives its source of energy mainly from oxidation of FAs. The oxidation of intra-hepatic FAs occurs primarily within mitochondria beta-oxidation and, to a much smaller extent, in peroxisomes and microsomes. Beta-oxidation requires FAs to be transported from the cytoplasm to the mitochondrial matrix, i.e., across the mitochondrial double membrane. This process needs the “activation” of FAs by co-enzyme A, which is accomplished by fatty acyl-CoA synthetase in the cytoplasm [45]. Then, carnitine palmitoyl transferase (CPT1) converts fatty acyl-CoA to fatty acyl carnitine in the outer mitochondrial membrane, which is eventually shuttled across the inner mitochondrial membrane by carnitine translocase [46]. Of great interest, targeting mitochondrial FAs oxidation through CPT1 in mouse models has been suggested as a strategy to treat obesity-related disorders including NAFLD [47]. Hepatic beta-oxidation/ketogenesis has been suggested to occur in patients with NAFLD [38,39,42,43]. Studies performed in rodent models have demonstrated that modulation (up- or down-regulation) of intra-hepatic oxidation of FAs by various stimuli influences IHTG content. Accordingly, genetic or experimentally-induced deficiency of hepatic enzymes involved in mitochondrial beta-oxidation may cause accumulation of IHTG [48], while an increase in the expression or activity of hepatic enzymes responsible of oxidation of FAs may decrease the accumulation of IHTG [49]. However, human studies have not led to consistent conclusions. In fact, while subjects with NAFLD have been reported to have hepatic mitochondrial structural and functional abnormalities (such as loss of mitochondrial cristae and paracrystalline inclusions [50,51], decreased mitochondrial respiratory chain activity [52], impaired ability to resynthesize ATP following a fructose challenge [53], and increased hepatic uncoupling protein 2 [54]), all of these abnormalities could affect the production of hepatic energy but not oxidation of FAs, possibly representing an adaptive uncoupling of oxidation of FAs and ATP production, which allows the liver to oxidize excessive fatty acid substrates, without generating unneeded ATP [51,52,53,54].

Hepatic secretion of triglycerides in the form of VLDL particles for delivery to peripheral tissues (including skeletal muscle, cardiac muscle and adipose tissue) is an important pathway for the mobilization of liver fat. The VLDL secretion rate appears to depend, not only on the availability of hepatic triglycerides, but also on the overall capacity for VLDL assembly. Hepatic VLDL assembly comprises two steps, both of which require the action of microsomal triglyceride transfer protein [55]. The first step involves the partial lipidation of a newly synthesized apolipoprotein (apo) B-100 molecule to form a small and dense VLDL precursor. In the second step, this small and dense precursor combines with a large triglyceride droplet to form a mature and triglyceride-rich VLDL, which is subsequently secreted into plasma [55,56,57]. Each VLDL particle contains one molecule of apoB100, which is required to export VLDL from the liver. The mechanisms responsible for the inadequate export of hepatic VLDL-triglyceride in patients with NAFLD are unknown. In such patients, the secretion rate of VLDL-triglyceride is much higher than that encountered in subjects without NAFLD [58], while VLDL particles (VLDL-apoB100) have been found to be either not different [58] or only slightly greater [59]. This suggests that VLDL particles produced by NAFLD patients may contain more triglycerides and may be larger than those produced by normal subjects. Animal studies have demonstrated that very large VLDL particles cannot be secreted from the liver because they exceed the diameter of the sinusoidal endothelial pores. Therefore, this inability can result in excessive IHTG accumulation, leading to hepatic steatosis [60]. In a very recent study, Fabbrini et al. [61] evaluated the physiologic mechanisms of weight gain-induced steatosis in 27 subjects. They found that weight gain increased the export of triglycerides from the liver by secreting a higher number of triglyceride-rich VLDL particles. However, this increase was not sufficient to fully compensate for the increased rate of IHTG production [61].

It is the only Food and Drugs Administration (FDA)-approved drug labelled for weight loss in children above 12 years of age [78]. Orlistat inhibits pancreatic lipase to induce fat malabsorption. Common side effects are abdominal cramps, flatulence (due to unabsorbed fat in the fecal mass), interference with absorption of fat-soluble vitamins [17,79,80], and chronic kidney disease (due to secondary hyperoxaluria) [81].

Omega-3 are polyunsaturated (PU) FAs that regulate transcription factors related to hepatic lipid metabolism, leading to increased FA oxidation and down-regulation of pro-inflammatory genes [82]. Omega-3 FAs have been reported to lower circulating triglycerides, by decreasing the hepatic secretion of VLDL cholesterol or by increasing chylomicron metabolism. The effect of omega-3 FAs on triglycerides chiefly involves the suppression of hepatic VLDL apoB production and apoB pool size. Chan et al. [83] showed that fish oil supplementation comprising 45% eicosapentaenoic acid and 39% docosahexaenoic acid (DHA) lowered triglycerides (−18%) and VLDL apo B (−20%) and the hepatic secretion of VLDL apo B (−29%) compared with placebo. The percentage of conversions of VLDL apo B to intermediate-density lipoprotein (IDL) apo B, VLDL apoB to low density lipoprotein (LDL) apoB, and IDL apoB to LDL apoB also increased significantly. This effect determines a 35% reduction in triglyceride synthesis and an increase in FA mitochondrial oxidation. In particular, omega-3 FAs induce the aggregation of apoB after its exit from the ER, but before it leaves the Golgi. Upon exit from the Golgi, this aggregate material becomes extensively oxidized, and converted into large aggregates. The aggregates slowly degrade by an autophagic process [84]. In a study using cultured hepatocytes, the effects of incubation with palmitic acid, oleic acid, and DHA on ER stress and apoB-100 secretion were compared [85]. The investigators found that both oleic acid and palmitic acid decrease the secretion of apoB-100 by inducing ER stress; palmitic acid was more potent, both in terms of dose and time of exposure, most likely due to its ability to increase the synthesis of ceramide. In contrast, DHA which was the most potent of the three FAs in terms of inhibiting apoB-100 secretion via stimulation of autophagy, did not induce ER stress. Furthermore, it has been shown that DHA may interfere with apoCIII gene transcription, thereby potentially decreasing the negative effect of apoCIII on LpL activity [86].

A few studies have investigated the effects of n-3 long chain-PUFAs on pediatric NAFLD [87,88]. Nobili et al. reported a short-term (six months) [87] and long-term (up to 24 months) [88] effects of DHA, after 6, 12, 18, and 24 months of treatment with different concentrations (DHA 250 mg/day and 500 mg/day) combined with diet and exercise. In these studies, algae DHA supplementation improved liver steatosis and was able to reduce the levels of serum alanine aminotransferase and triglycerides. More recently, Nobili et al. found that treatment with DHA (250 mg/day) for 18 months improved histopathological parameters such as hepatocyte steatosis, ballooning, and NAFLD activity score but was ineffective on fibrosis [89]. Pacifico et al. investigated the effect of six months of DHA on liver fat in 58 children with biopsy-proven NAFLD [90]. Hepatic fat fraction as measured by magnetic resonance imaging decreased significantly in the group receiving DHA in comparison with that receiving placebo.

The FDA has recently approved ezetimibe use for treatment of hypercholesterolemia in children above 10 years. Ezetimibe selectively inhibits cholesterol absorption from intestine by binding to the brush border, and consistently lowers LDL-cholesterol [91]. Ezetimibe has also been shown to decrease hepatic lipid synthesis and favor lipid removal from the liver by preventing the degradation of microsomal triglyceride transfer protein, with a consequent inhibition of the development of NAFLD in animal models [92,93]. These results suggest that ezetimibe can increase lipoprotein assembly in the liver and perhaps in the intestine. Studies in adult patients with NAFLD have shown improvement in hepatic histology but with worsening or no effects on insulin sensitivity [94,95]. Promising results have been demonstrated when ezetimibe has been used in combination with statins [92]. No studies are available in children with NAFLD.

Statins are specific inhibitors of 3-hydroxy-3methylglutaryl coenzyme A reductase, the rate-limiting enzyme in cholesterol biosynthesis [96]. They also promote the synthesis of LDL-receptor (LDL-r) acting on the LDL-r gene, ultimately increasing the expression of membrane LDL-r [91]. In addition to their cholesterol-lowering effects, statins also possess pleiotropic properties that account for their anti-inflammatory, anti-proliferative, anti-thrombotic, anti-oxidative, anti-cancer, and immuno- modulatory actions in vitro and in vivo [97]. Statins can reduce liver triglycerides [98] and ameliorate severe hepatic steatosis [99]. The American Association for the Study of Liver Disease Guidelines recommends that statins can be used for treatment of dyslipidemia in patients with NAFLD and NASH [100]. Statins are now considered as a first line pharmacological intervention for pediatric patients with severe dyslipidemias [101]. However, no data are available in pediatric NAFLD.

The farnesoid X receptor (FXR) is a bile acid-activated nuclear receptor. When bound to the FXR, lipophilic bile acids improve insulin sensitivity and decrease hepatic gluconeogenesis and circulating triglycerides [102]. These effects are mediated by decreased hepatic lipid synthesis and increased peripheral clearance of VLDL [103,104,105]. Thus, targeting FXR may offer new perspectives for the treatment of NAFLD. Support comes from studies investigating the effects of FXR activation by 6-ethyl-chenodeoxycholic acid, a potent activator of FXR, in rats with diabetes mellitus, obesity, insulin resistance, and liver steatosis [106]. FXR activation protected against body weight gain and liver and muscle fat deposition, and reversed insulin resistance. Activation of FXR reduced liver expression of genes involved in FA synthesis, lipogenesis and gluconeogenesis. Preliminary data in adult patients with NAFLD treated with 6-ethyl-chenodeoxycholic acid (obeticholic acid) have shown promising results [107], but its long-term benefits and safety need clarification. There are no data in pediatric populations.