Non-alcoholic fatty liver disease (NAFLD) is a clinicopathological syndrome, which encloses a spectrum ranging from pure steatosis to steatohepatitis, fibrosis and cirrhosis eventually with scarce steatosis [1–4], while the non-alcoholic steatohepatitis (NASH) definition relies 4on characteristic histological findings similar to those of alcoholic hepatitis . Both situations are defined by exclusion of alcohol consumption of more than 20 g/day .
The NAFLD frequency is increasing, now being recognized as one of the most frequent causes of hepatic disease , occurring in 20% of the general population, whereas NASH occurs in about 3% [8–10].
Primary NAFLD must be differentiated from secondary steatosis or steatohepatitis, since their pathogeneses and prognosis are different, with a worse outcome in the latter . NAFLD may be secondary to nutritional, drug, genetic and environmental causes, among others.
Many authors consider primary NAFLD as the hepatic manifestation of the insulin resistance (IR) syndrome [12–14]. IR is associated with hyperinsulinism, glucose intolerance and type 2 diabetes mellitus, hypertriglyceridaemia and low levels of high-density lipoproteins, hypertension, fibrinolysis, accumulation of visceral fat, hyperuricaemia and polycystic ovarian syndrome . Closely associated with IR is a constellation of manifestations now defined as the metabolic syndrome on the basis of three or more criteria out of five defined by the Third Report of the National Cholesterol Education Program Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults (ATP III): waist circumference, glucose, high-density lipoprotein-cholesterol, triglycerides, and arterial pressure .
NASH shows a strong association with the features of the metabolic syndrome, namely obesity (which is present in more than one-half of the patients, reaching 90% in some series), dyslipidaemia (in 21–92%) and type 2 diabetes mellitus (in 20–55%) [7,17–20]. In fact, the Verona Diabetes Study, with 7148 patients, showed a higher increase in the liver disease-related mortality than in the cardiovascular mortality of patients with diabetes mellitus, when compared with the general population .
Frequency of insulin resistance in NAFLD
There is discrepancy in the literature concerning the real frequency of IR in NAFLD, probably depending on the different criteria used, and on whether the all NAFLD spectrum or only NASH is considered. In fact, some authors [12,13] evaluated IR using the homeostatic model assessment (HOMA), while others used the frequently sampled intravenous glucose tolerance test . Furthermore, Chitturi et al. considered IR if HOMA-IR>1.64 , while Marchesini et al. considered IR if HOMA-IR≥3 . Concerning the definition of the metabolic syndrome, different criteria were also used, such as the World Health Organization definition  or the ATP III . Although insulin sensitivity is a continuum in the general population, and therefore any attempt to define a cut-off for IR is arbitrary, uniform criteria should be used in future prospective studies of NAFLD and IR; HOMA>3 as criteria of IR and the ATP III criteria for defining the metabolic syndrome are probably the more consensual.
Taking into account these discrepancies, IR is very frequent in NAFLD patients (47–98%), even in the absence of diabetes mellitus [13,22,24]. However, despite NAFLD association with the features of the metabolic syndrome, only 36% of patients with NAFLD fulfil at least three criteria and thus can be classified as having that syndrome, which was associated with a higher risk of having NASH and severe fibrosis . It should be noticed that the prevalence of the metabolic syndrome as defined by the ATP criteria in the general adult population from the United States is about 22% . Therefore, although there is a strong association, there are still a percentage of patients that do not have evidence of IR at the time of diagnosis. This can be due either to the fact that there are other causes of secondary NASH not identifiable at the moment, or to the fact that in some patients liver disease precedes IR, or yet that the indirect methods used to evaluate IR such as the HOMA are not sensitive enough.
In the present issue, Sequeira et al. report IR to be present in one-third of patients with NAFLD (note that most series relate IR with NASH and not with NAFLD), which is much lower than usually described. Nevertheless, their population included primary as well as secondary NAFLD. In fact, in patients who had exclusively metabolic risk factors, IR occurred in 50%, in agreement with the previous literature. On the other hand, only 9.4% of patients exposed to chemicals with no other risk factor had IR. Diagnosis of the metabolic syndrome (at least three criteria present) was present in 29.7% of cases of NAFLD, less frequent than previously reported in the literature, probably because the series included primary and secondary NAFLD. It is also interesting to note that, in conformity with previous literature, IR was associated with advanced fibrosis, which suggests that IR is important not only in the development of NAFLD but also in the progression of the disease. These results confirm the association of primary NAFLD with IR, underscoring that this is probably not the case in forms of secondary NAFLD.
NAFLD/NASH and IR association is relevant, as they are linked in terms of pathogenesis. NASH pathogenesis has been interpreted as the result of two hits, the first leading to steatosis and the second to inflammation and necrosis . Recently it was suggested that a single hit, IR, could be enough to explain the whole spectrum of NAFLD . In fact, a four-step model was recently proposed in which the first step is steatosis facilitated by insulin, the second is necrosis induced by intracellular lipid toxicity or lipid peroxidation, the third is release of bulk lipid from hepatocytes into the interstitium leading to direct and inflammatory injury to hepatic veins, and the fourth step is venous obstruction with secondary collapse and, ultimately, fibrous septation and cirrhosis .
IR courses with resistance to some insulin actions and a compensatory increase of insulin levels. As a result, many metabolic pathways may be over-activated. In peripheral tissues, IR leads to a decrease of the insulin inhibitor effect on hormone-sensitive lipase, which remains active, enhancing triacylglycerol hydrolysis to glycerol and fatty acids, increasing free fatty acids in the plasma and reaching the liver. In the liver, IR leads to an increase of fatty acid oxidation and gluconeogenesis. On the other hand, hyperinsulinism leads to an increase of fatty acid synthesis and a decrease of triglycerides output as very low density lipoproteins. In conclusion, IR/hyperinsulinism is associated with triglyceride accumulation in the liver, and thus steatosis, as a result of an increase of the input and synthesis of fatty acids and a decrease in the output of triglycerides. Adipose tissue, and particularly visceral adipose tissue, has a very important role in the development of IR and NAFLD. Adipose tissue is now recognized as an endocrine organ and cytokine producer. It is able to produce tumour necrosis factor-alpha (TNF-α) and three hormones: leptin, adiponectin and resistin. TNF-α, leptin and resistin (the latter still controversial) further increase IR [29–31]. Adiponectin has opposite effects when compared with TNF-α, being protective against IR . As suggested by animal models, there is a self-perpetuating pathway between IR and inflammation. It was demonstrated in the murine model of diet-induced steatohepatitis that free fatty acids markedly stimulated TNF-α expression in an NF-κβ-dependent process because a super-repressor of I-kB blocked TNF-α up-regulation. TNF-α can promote IR by signalling IKK-β activation and c-jun-N-terminal kinase activation [33,34].
There was previous evidence of increased cytochrome P450 2E1 (CYP2E1) expression being implicated in the development of NAFLD . There is now recent evidence that IR and CYP2E1 expression may be interrelated through the ability of CYP2E1-induced oxidant stress to impair hepatic insulin signalling. It was shown in the methionine and choline-deficient diet mouse model of steatohepatitis with CYP2E1 overexpression that insulin-induced IRS-1, IRS-2 and Akt phosphorylation were similarly decreased. This inhibition of insulin signalling by CYP2E1 overexpression was partially c-jun-N-terminal kinase dependent. These findings indicate that increased hepatocyte CYP2E1 expression and the presence of steatohepatitis result in the down-regulation of insulin signalling, potentially contributing to the IR associated with NAFLD .
No treatment has scientifically proved to ameliorate NAFLD lesions or to avoid its progression. The various therapeutic alternatives are aimed at interfering with the risk factors involved in the pathogenesis of NASH, in order to prevent the progression to end-stage disease associated with the development of cirrhosis and liver failure.
Because of the importance of IR in the basis of NAFLD pathogenesis, it is easy to understand the importance of increasing insulin sensitivity to treat these patients. To do so, the most important therapeutic measure is an attempt to change lifestyle mostly by dieting and implementing physical activities in order to lose weight, when overweight or obesity are present. This seems to lead to an improvement in biochemical tests and in steatosis, although no benefit in terms of inflammation or fibrosis has been demonstrated . These benefits are coupled with an improvement in glucose tolerance.
In what concerns drugs, insulin-sensitizing agents are the more promising. The most used agents are metformin [38,39] and the thiazolidinediones such as pioglitazone  and rosiglitazone . They proved to be effective in reducing IR and aminotransferase levels with an improvement in hepatic histology in the short period. Unfortunately, the long-term response is still unknown and there are no controlled studies with these agents.
Conflict of interest
Both authors contributed to the writing of the paper. H.C.-P. also reviewed the text.
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