As far as we are aware, this is the first RCT to examine the potential beneficial effects of HP-diet, β-cryptoxanthin, or both in NAFLD. Findings suggest that a hypocaloric HP-diet supplemented with β-cryptoxanthin leads to more significant improvements in liver enzymes and hepatic steatosis in patients with NAFLD over 12 weeks, compared to a standard hypocaloric diet. Moreover, the potential safety of this treatment regimen for patients with NAFLD is indicated by the relatively low rate (i.e., 21.7%) and benign nature (i.e., mild and self-limiting gastrointestinal discomfort or headache) of the reported adverse events as well as the extremely low drop-out rate (i.e., 4.3%) in the HP-diet & β-cryptoxanthin group.
In this study, patients in the HP-diet & β-cryptoxanthin and HP-diet groups experienced greater reductions in weight than those in the β-cryptoxanthin and control groups at the end of the study. These observations are consistent with the existing evidence indicating that, in a negative energy balance state, HP-diets are more efficacious in weight reduction as compared to NP-diets [33, 34]. Even though the exact mechanisms are not well-determined, this could be justified by the fact that HP-diets are less calorically efficient than NP-diets (i.e., dietary proteins require more energy to be metabolized than other macronutrients do) and lead to more a significant increase in thermogenesis [33, 34].
Because obesity plays a pivotal role in the development and pathogenesis of NAFLD, lifestyle modifications targeted at achieving a weight reduction of at least 3–5% of normal weight represent the first-line of treatment for affected patients [2, 8, 9]. In this respect, findings of a systematic review of 23 interventional studies examining the therapeutic properties of lifestyle interventions (e.g., a hypocaloric diet) in NAFLD indicate that lifestyle modifications leading to weight loss result in significant reduction of circulating liver aminotransferase levels and/or hepatic fat concentration [35]. In line with these findings, following a hypocaloric diet by our patients for 12 weeks led to significant reduction of serum levels of liver enzymes and grade of hepatic steatosis in all study groups. Since patients in the HP-diet & β-cryptoxanthin, HP-diet, β-cryptoxanthin, and control groups respectively lost an average of 6.8%, 6.9%, 3.9%, and 3.9% of their baseline weight in 12 weeks, the observed improvements of NAFLD in all study groups and especially in the control group could thus be largely attributed to the weight-reducing effects of prescribed hypocaloric diets.
As this is the first RCT to assess the effects of HP-diet and/or β-cryptoxanthin in NAFLD, the direct comparison of our findings with those from similar studies is not feasible at the moment. However, greater improvements in NAFLD in the HP-diet & β-cryptoxanthin group as compared to the control group in this study is in line with findings of a few quasi-experimental studies in humans indicating that the prescription of a hypocaloric HP-diet (providing 35–47% of daily energy intake as protein) for 2–11 weeks in NAFLD patients significantly reduces hepatic fat concentration and/or circulating liver enzyme levels [19–22]. Furthermore, our observations are in accordance with a substantial body of evidence from experimental animal research suggesting that HP-diets with or without calorie restriction significantly improve hepatic steatosis in animal models of NAFLD [13–18, 23]. Moreover, results of the present study are supported by those from a couple of experimental works in animals indicating that administration of a normocaloric diet with 0.003% β-cryptoxanthin to mice with diet-induced non-alcoholic steatohepatitis for 12 weeks leads to significant improvement of hepatic steatosis and circulating liver aminotransferase levels [24, 25].
Although the greater efficacy of a hypocaloric HP-diet supplemented with β-cryptoxanthin in improvement of NAFLD as compared to a standard hypocaloric diet in our study could be partly attributed to the higher weight reduction observed in the HP-diet & β-cryptoxanthin group, it is logical to assume that there might be other mechanisms involved. This assumption is in fact supported by the results of a quasi-experimental study by Bezerra-Duarte et al., in which a hypocaloric HP-diet significantly reduced serum levels of liver enzymes in NAFLD patients in the absence of any significant weight reduction [20]. It is noteworthy that a host of potential mechanistic pathways have been proposed to explain the therapeutic properties of HP-diets in NAFLD, among most important of which are induction of lipolysis and lipid utilization as well as suppression of lipogenesis, cell stress, and inflammation in the liver [13, 14]. In addition, a few mechanisms have recently been identified by which β-cryptoxanthin supplementation might exert some beneficial effects in NAFLD [12]. In fact, mechanistic animal studies suggest that β-cryptoxanthin could suppress insulin resistance, oxidative stress, inflammation, and macrophages/Kupffer cells activation, all of which play central roles in the development and/or progression of NAFLD [12, 24, 25]. At last, it is also possible that combination of a hypocaloric HP-diet and β-cryptoxanthin supplementation in the HP-diet & β-cryptoxanthin group of this study have acted synergistically through all or a few of the aforementioned mechanisms to cause more significant improvements in NAFLD.
Some points need to be considered when interpreting the findings of the present study. First, although recent evidence support the diagnostic accuracy and reliability of US for the detection of moderate-to-severe hepatic steatosis as compared to histology, the gold standard method for NAFLD diagnosis and grading is still liver biopsy [2, 3, 36]. Second, despite the fact that 80.4% of all patients guessed wrong about their allocated intervention, it must be noted that due to the nature of prescribed diets it was not possible to achieve complete blinding (e.g., patients in the HP-diet & β-cryptoxanthin and HP-diet groups could have detected that they were consuming more amounts of protein than they normally did). Third, even though our ANCOVA models were adjusted for several potential confounders, the probability of residual confounding bias because of unknown or unmeasured confounding variables (e.g., genetic determinants of NAFLD including the palatin-like phospholipase domain-containing 3 or the transmembrane 6 superfamily member 2 gene polymorphism) cannot be entirely ruled out [3].