- freely available
Nutrients 2013, 5(5), 1544-1560; doi:10.3390/nu5051544
Abstract: Non-alcoholic fatty liver disease is marked by hepatic fat accumulation not due to alcohol abuse. Several studies have demonstrated that NAFLD is associated with insulin resistance leading to a resistance in the antilipolytic effect of insulin in the adipose tissue with an increase of free fatty acids (FFAs). The increase of FFAs induces mitochondrial dysfunction and development of lipotoxicity. Moreover, in subjects with NAFLD, ectopic fat also accumulates as cardiac and pancreatic fat. In this review we analyzed the mechanisms that relate NAFLD with metabolic syndrome and dyslipidemia and its association with the development and progression of cardiovascular disease.
Non-alcoholic fatty liver disease (NAFLD) has been considered a benign disease often associated with central obesity and insulin resistance and in general with factors of the metabolic syndrome (Figure 1). However, recent studies have highlighted that NAFLD is a chronic condition, ranging from benign steatosis, (i.e., hepatic triglyceride accumulation >5.5% using magnetic resonance imaging [1,2] or >5% corresponding to 50 mg/g by wet weight), to more significant liver injury including lobular inflammation, hepatocyte ballooning, fibrosis and cirrhosis, i.e., non-alcoholic steato-hepatitis (NASH) .
Excess liver fat is extremely common and prevalence of NAFLD has been increasing mainly because of the increased prevalence of obesity. It has been estimated that as many as 30% of adults in USA and other Western countries have NAFLD . The real prevalence is unknown since NAFLD is often undiagnosed and most subjects with NAFLD, even those with diabetes, have normal liver aminotransferases and clinicians do not suspect the potential presence of NAFLD [5,6,7]. NAFLD is more prevalent among obese subjects and also in patients with type 2 diabetes independently of degree of obesity . The prevalence increases to 57% in obese subjects, 70% in diabetic subjects and 90% in morbidly obese people. On the other hand NASH may be present in up to 3% of the general population and in up to two thirds of individuals with morbid obesity and/or type 2 diabetes [4,8]. In addition to hepatic complications, patients with NAFLD are at increased risk for cardio-metabolic complications such as type 2 diabetes (T2DM) and cardiovascular disease (CVD) [4,9].
In this article we have reviewed the current literature trying to highlight the mechanisms that are responsible for the development of NAFLD and that are at the base for the increased cardiometabolic risk in patients with NAFLD.
2. Insulin Resistance and the Development of NAFLD
Several studies have highlighted that insulin resistance is a characteristic feature of NAFLD [10,11,12], even when subjects are not obese . However, NAFLD per se cannot be considered a cause for insulin resistance but rather a consequence as shown by studies in subjects genetically predisposed to NAFLD. In fact subjects with either mutation for PNPLA3 gene [14,15], familial hypobetalipoproteinemia [16,17] or mutation in DGAT [18,19], have fatty liver but peripheral and hepatic insulin sensitivity comparable to matched subjects without mutation and NAFLD (Figure 1). On the other hand NAFLD is highly prevalent among patients with type 2 diabetes (up to 70%)  that show increased hepatic triglyceride accumulation independently of BMI .
Insulin resistant subjects with NAFLD show reduced insulin sensitivity not only at the level of the muscle but also at the level of the liver and adipose tissue [7,13,21]. In insulin-resistant conditions, the adipose tissue becomes resistant to the antilipolytic effect of insulin and the release of fatty acids is increased . Insulin resistance is accompanied by increased insulin levels that, in the presence of increased lipolysis and/or increased fat intake, promote hepatic triglyceride synthesis . Adipose tissue insulin resistance is quantified using the index Adipo-IR (FFA × INS) [7,23] that reflects the inability of insulin to suppress peripheral lipolysis. In subjects with NAFLD, even if not obese, FFA concentrations and Adipo-IR are increased compared to control subjects [13,24], despite an increase in both hepatic and systemic lipid oxidation , and in VLDL-TG secretion [25,26]. Adipo-IR is also a marker of hepatic liver injury .
Under postprandial conditions, an important source of FFA is due to the increased spillover from chylomicrons . The increased spillover reflects the inefficiency in dietary fat storage and results in excess FFA. FFA are taken up by organs saturating their oxidative capacity  and accumulated as ectopic fat, mainly as intramyocellular and hepatic lipids [28,29] but also as cardiac and pancreatic fat. It has been hypothesized that ectopic fat could be a defense mechanism against lipotoxicity [30,31] and that subjects with NAFLD develop NASH and cirrhosis only in consequence of a second hit due to increased inflammation and reactive oxygen species .
3. Impact of Hepatic and Visceral Fat Accumulation on Metabolic Profile
Hepatic (IH-TG) and visceral fat (VF) are highly correlated [7,32] but independently predict the presence of metabolic alterations [12,33]. The direct impact of VF on glucose and lipid metabolism is however controversial and difficult to prove since it is a small depot compared to subcutaneous fat . Although a correlation is found between peripheral glucose clearance and IH-TG or VF [7,13,35], it is unlikely that IH-TG and VF contribute directly through FFA release and lipotoxicity to muscle insulin resistance. Although VF accounts for as much as 38% of total fat in some extreme cases , it is unlikely that FFAs released by VF are responsible for muscular lipotoxicity , except through cytokines released by the dysfunctional tissues.
Previous studies indicate that hepatic fat and not VF was associated with insulin resistance. When subjects with different IH-TG content were matched on similar VF, they showed increased hepatic and peripheral insulin resistance and increased VLDL-TG secretion rate but no difference was observed between subjects matched for IH-TG but different VF  (Figure 2). Moreover, the partial VF reduction by omentectomy did not further improve peripheral and hepatic insulin sensitivity due to weight loss after surgery . However, this type of analysis can be misleading, since subjects with normal IH-TG had high visceral fat content and subjects with matched IH-TG had on average 13% steatosis (Table 1). We therefore analyzed subjects with low VF and low IH-TG vs. those with both high VF and IH-TG, finding that insulin resistance increases proportionally to both visceral and liver fat (Figure 2D–F). By performing a simple correlation between hepatic and visceral fat and indexes of insulin resistance we observed that visceral and hepatic fat had a similar correlation  (Figure 3).
|Group of subjects||IHTG (%)||VF||Reference|
|Matched on VAT||Normal IHTG||3.6 ± 0.5||1.29 ± 0.24 kg|||
|High IHTG||25.3 ± 3.5||1.34 ± 0.18 kg|
|Matched on IHTG||Low VF||13.2 ± 3.5||0.76 ± 0.08 kg|||
|High VF||13.2 ± 3.3||1.94 ± 0.32 kg|
|Low IH-TG||Low VF||2.1 ± 0.6||70 ± 8 cm2|||
|High VF||12.9 ± 1.9||84 ± 5 cm2|
|High IH-TG||Low VF||3.2 ± 1.6||165 ± 41 cm2|||
|High VF||23.3 ± 1.7||159 ± 9 cm2|
The analysis reported in Figure 2, Figure 3 shows that IH-TG and VF contribute similarly to the increase in insulin resistance at all levels, liver, muscle and adipose tissue. In conditions of insulin resistance hepatic insulin sensitivity and hepatic insulin clearance are decreased proportionally to both hepatic and visceral fat content (Figure 3) and this is more evident in patients with Type 2 diabetes compared with non-diabetic subjects [7,35]. The relative contribution of IH-TG and VF to IR is in agreement with previous studies that have shown that obese subjects with increased VF have increased whole-body lipolysis compared to lean subjects [38,39] and subjects with NAFLD have preferential accumulation of abdominal fat. In upper body obesity, portal FFA concentrations, resulting from both systemic and VF lipolysis, is significantly greater than arterial FFA concentrations, exposing the liver to even greater amounts of FFA [40,41].
In the fasting state the liver is the main site of glucose production through gluconeogenesis (GNG) and glycogenolysis . The contribution of GNG is increased in insulin resistant subjects  but because of increased circulating insulin, glycogenolysis is diminished and therefore hepatic glucose production remains within normal ranges, because of a mechanism named hepatic autoregulation [41,43]. Only when hepatic autoregulation is lost, i.e., in T2DM, both GNG and glycogenolysis are increased and subjects develop hyperglycemia . Subjects with NAFLD, despite high circulating insulin levels, have reduced hepatic insulin sensitivity and postprandial glucose clearance and increased free fatty acid and triglyceride concentrations [7,13,21,44]. Recently, Sunny et al. have shown an increase in GNG in a small group of subjects with NAFLD without known T2DM compared with controls . We have found different results in a large group of T2DM patients: GNG was increased in T2DM vs. controls, but not associated with liver fat content . On the other hand we have observed a direct relationship between GNG and VF and fasting hyperglycemia, while glycogenolysis was not correlated to either VF or IH-TG [7,46]. Liver fat in this study ranged from 0% to 52%, while in the paper by Sunny et al.  it ranged from 0% to 21% and this could explain at least in part the different results. Another difference could be the relative contribution of lipolysis and FFA to GNG. It is well established that elevated free fatty acids (FFA) stimulate hepatic GNG  and VLDL-TG production in the face of hyperinsulinemia . Despite the differences in the type of NAFLD subjects (with or without T2DM) and the degree of liver steatosis, the lack of correlation between GNG and hepatic TG shows that not all subjects with NAFLD have increased GNG and indicates that hepatic TG do not participate directly to increase GNG. On the other hand the two pathways are independent and with different turnover rates, GNG is a dynamic process  while hepatic TG are not changing rapidly, e.g., after the intake of a high-fat meal change in hepatic TG was not significant . Thus, we have hypothesized that in conditions of insulin resistance the increased lipolysis (especially from VF) generates an overload of glycerol and FFA to the liver that are “cleared” through increased TG synthesis and GNG (where glycerol is used as a gluconeogenic substrate and FFA provide the ATP necessary for the GNG process through hepatic beta-oxidation). Indeed, diabetic hyperglycemia is proportional to increased visceral fat content and GNG .
4. Fatty Liver, Dyslipidemia and the Metabolic Syndrome
The presence of dyslipidemia (hypercholesterolemia, hypertriglyceridemia, or both) has been reported in 20% to 80% of cases associated with NAFLD . Liver fat content reflects the equilibrium between FFA flux through lipolysis, fatty acid oxidation, de-novo lipogenesis and VLDL secretion (Figure 4). The hepatic triglyceride accumulation is probably a consequence of saturation of fatty acid oxidation and VLDL secretion (Figure 4) since both these pathways are up-regulated rather than decreased in patients with NAFLD [13,25,26] and abnormality in apoB secretion has been excluded .
Postprandial hyperlipidemia and FFA spillover from chylomicrons worsen the situation [27,50]. Chylomicrons and triglyceride-rich lipoproteins can contribute either directly to plaque formation, following penetration of the arterial wall at sites of enhanced endothelial permeability, or potentially indirectly following liberation of lipolytic products (such as FFA and lysolecithin) which may activate pro-inflammatory signalling pathways in endothelial cells .
In patients with NAFLD adiponectin concentrations are decreased compared to normal subjects [52,53], despite higher lipolysis and fatty acid concentrations associated with increased hepatic and systemic fatty acid oxidation. It is well established that adiponectin activates AMPK and peroxisome proliferator-activated receptor (PPAR)-α, thus stimulating fatty acid oxidation in liver and muscle . Adiponectin levels also correlate inversely with plasma TGs, positively with HDL-cholesterol levels and LDL size, suggesting a role in lipoprotein metabolism [26,55]. Thus, low adiponectin levels in NAFLD can be seen as a limit in the capacity of further increase lipid oxidation in response to FFA overload, redirecting FFA towards re-esterification.
Fatty Liver and Metabolic Syndrome
It is not surprising that many studies have highlighted the association between NAFLD and several factors of metabolic syndrome, especially abdominal obesity, insulin resistance, increased serum triglycerides and small dense LDL and low HDL [7,25,26,44,56,57,58,59,60] (Figure 4). It has also been proposed that NAFLD could be considered the hepatic manifestation of metabolic syndrome [61,62]. Prevalence of the metabolic syndrome in NAFLD has been estimated to vary from 18% in normal-weight to 67% in obese subjects [61,62,63,64]. Moreover, liver fat accumulation is very common in type 2 diabetes  and a strong link has been observed between abdominal ectopic fat accumulation and the development of hypertension [62,65,66]. There are plenty of data linking the liver enzymes ALT and GGT (both correlated with liver fat) with incidence of diabetes . It has been shown that up to 85% of subjects with NAFLD compared to 30% in controls are insulin resistant and have abnormal glucose metabolism, i.e., prediabetes or T2DM, of which they were unaware . This could explain the increased CVD risk that is often observed in patients with NAFLD highlighted by several epidemiological reports [9,69].
5. NAFLD and Cardiovascular Disease
NAFLD is associated with increased CVD [9,60,70]. The relationship between CVD and NAFLD (diagnosed with either ultrasound or liver biopsy) was evaluated in studies with limited number of subjects . Thus, larger longitudinal studies are needed to demonstrate if NAFLD is a primary cause of CVD and/or if NAFLD per se increases the risk of CVD mortality. Several factors that can explain the increased CVD risk in subjects with NAFLD as already discussed in the previous paragraphs. Among these there are the increased lipolysis and VLDL secretion [13,25], the atherogenic lipoprotein profile, i.e., increased small dense LDL fractions and reduced in HDL fractions [7,44,59,60], the hyperglycemia due to hepatic overproduction of glucose, the increased release of inflammatory factors such as fibrinogen and C reactive protein (CRP) [7,13,71]. Besides quantitative reduction in HDL concentration, also qualitative alterations occur , which may substantially contribute to the atherogenic risk of NAFLD.
Fat accumulation in the liver and oxidative stress induce the secretion of inflammatory markers such as, IL-6, TNF-α, Fetuin-A, CRP, and fibrinogen. Fetuin-A is a protein secreted by the liver, it is a natural inhibitor of the insulin receptor tyrosine kinase  and an endogenous ligand for TLR4 through which lipids induce insulin resistance . It has been shown that Fetuin-A induces low grade inflammation [73,74], is associated with endothelial dysfunction , carotid atherosclerosis  and an increased risk of myocardial infarction (MI) and ischemic stroke (IS)  and type 2 diabetes . Increased CRP promotes inflammation and accelerates atherosclerosis by increasing the expression of plasminogen activator inhibitor-1 and adhesion molecules in endothelial cells, inhibiting nitric oxide formation and increasing LDL uptake into macrophages . All these metabolic abnormalities, common in subjects with NAFLD, have been shown to directly or indirectly promote atherosclerosis as confirmed by studies that showed increased carotid intima media thickness (IMT) and coronary atherosclerosis [9,44,60,69,71]. NAFLD is also associated with endothelial dysfunction and coronary artery disease [44,69,79,80,81].
The real prevalence of CV events in patients with NAFLD is still not known and probably underestimated. NAFLD is often not diagnosed since in the great majority of NAFLD subjects hepatic enzymes are within normal ranges and ultrasound technique is unable to detect NAFLD when fat infiltration is below 30% [82,83].
5.1. Fatty Liver and Atherosclerosis
Several studies have highlighted the association between NAFLD and increased carotid and coronary atherosclerosis [9,44,60]. In a large group of South Korean subjects, Sung et al. found that having fatty liver increases the risk of having coronary calcification and to develop type 2 diabetes . In a Japanese cohort, age, obesity (body mass index BMI ≥ 25 kg·m−2), hypertriglyceridemia and, to a lesser extent, hypertension were among the variables that predicted development of fatty liver . Previously it has been shown that hypertriglyceridemia is present in up to 64%  and indeed triglyceride and gamma glutamyl transferase (γGT) concentration, waist circumference and BMI are among the best predictors of fatty liver disease and related co-morbidities [44,85]. The RISC study showed that subjects with NAFLD are more prone to early carotid atherosclerosis even in the absence of metabolic syndrome and confounding diseases (hypertension, diabetes, cardiovascular diseases and dyslipidemia) . The RISC study also documented the relationship between fatty liver and the presence of early plaques at carotid bifurcation, as well as the associations between carotid plaque presence and established atherosclerotic risk factors, family history of cardiovascular disease (FH-CVD) or diabetes, insulin sensitivity, serum liver enzymes, adipokines, free fatty acids and high-sensitivity C-reactive protein (hsCRP) [44,80].
5.2. Fatty Liver and Endothelial Dysfunction
Patients with NAFLD have endothelial dysfunction and a significant decrease in brachial artery endothelial flow-mediated vasodilatation compared to the healthy controls [20,79,86]. This decrease is correlated to histological features of NAFLD independent of age, sex, BMI, HOMA-insulin resistance, and other metabolic syndrome (MS) components [20,79]. The integrity and the maintenance of the endothelium wall are important in protecting against atherosclerotic vascular disease. The regeneration of the endothelial monolayer, when it is damaged, is performed by circulating bone marrow derived-endothelial progenitor cells (EPCs) and so the concentration of these in the plasma reflects the endothelial repair capacity . Subjects with NAFLD have decreased plasma levels of EPCs that are correlated with arterial stiffening and endothelial dysfunction .
5.3. Fatty Liver and Coronary Artery Disease (CAD)
The presence of fatty liver is strongly associated with increased CAD risk [35,89] and CAD is a major cause of death in patients with NAFLD. This could also be explained by the fact that hepatic fat is often associated to cardiac fat and increased insulin resistance in these patients affects not only the liver but also other tissues like the heart . The RISC study has shown that fatty liver is associated with an increased 10-year coronary heart disease risk score even in subjects without diabetes and hypertension, i.e., at low risk for CVD [44,80]. Moreover patients with NAFLD, even without metabolic syndrome, have more vulnerable coronary soft plaques than healthy controls . In a large cohort of Taiwan workers, Lin et al. showed that patients with NAFLD were more likely to have CAD compared to patients without NAFLD, independent of obesity and other risk factors . In patients with T2DM Targher et al. showed a higher prevalence of coronary, cerebrovascular, and peripheral vascular disease increased in those with NAFLD as compared to those without NAFLD . However, despite the strong relationship between metabolic syndrome and CAD, it has been shown that some parameters of metabolic syndrome, like diabetes and hypertension, were better independent predictors of CAD than metabolic syndrome itself and that the association between NAFLD and CAD was independent of other demographic and metabolic factors .
In summary, NAFLD is associated with features of metabolic syndrome and is more prevalent among obese subjects and patients with type 2 diabetes independent of degree of obesity. The increased risk for cardio-metabolic diseases in NAFLD is caused by different factors among which hepatic overproduction of glucose, VLDL, inflammatory factors, C-reactive protein (CRP), and coagulation factors and by the presence of insulin resistance. Large trials that investigate the incidence of CVD and related mortality in subjects with NAFLD are needed to confirm this observation.
The research leading to these results has received funding from the European Community’s Seventh Framework Programme (FP7/2007-2013) under grant agreement n° HEALTH-F2-2009-241762 for the project FLIP. AG was also supported by a grant from the European Foundation for the Study of Diabetes (EFSD), and internal funds from the Italian National Research Council (CNR).
Conflict of Interest
The authors declare no conflict of interest for this work.
- Szczepaniak, L.S.; Babcock, E.E.; Schick, F.; Dobbins, R.L.; Garg, A.; Burns, D.K.; McGarry, J.D.; Stein, D.T. Measurement of intracellular triglyceride stores by H spectroscopy: validation in vivo. Am. J. Physiol. 1999, 276, E977–E989. [Google Scholar]
- Browning, J.D.; Szczepaniak, L.S.; Dobbins, R.; Nuremberg, P.; Horton, J.D.; Cohen, J.C.; Grundy, S.M.; Hobbs, H.H. Prevalence of hepatic steatosis in an urban population in the United States: impact of ethnicity. Hepatology 2004, 40, 1387–1395. [Google Scholar] [CrossRef]
- Day, C.P. Pathogenesis of steatohepatitis. Best Pract. Res. Clin. Gastroenterol. 2002, 16, 663–678. [Google Scholar] [CrossRef]
- Chalasani, N.; Younossi, Z.; Lavine, J.E.; Diehl, A.M.; Brunt, E.M.; Cusi, K.; Charlton, M.; Sanyal, A.J. The diagnosis and management of non-alcoholic fatty liver disease: Practice guideline by the American Gastroenterological Association, American Association for the Study of Liver Diseases, and American College of Gastroenterology. Gastroenterology 2012, 142, 1592–1609. [Google Scholar] [CrossRef]
- Fracanzani, A.L.; Valenti, L.; Bugianesi, E.; Andreoletti, M.; Colli, A.; Vanni, E.; Bertelli, C.; Fatta, E.; Bignamini, D.; Marchesini, G.; et al. Risk of severe liver disease in nonalcoholic fatty liver disease with normal aminotransferase levels: A role for insulin resistance and diabetes. Hepatology 2008, 48, 792–798. [Google Scholar]
- Kotronen, A.; Juurinen, L.; Hakkarainen, A.; Westerbacka, J.; Corner, A.; Bergholm, R.; Yki-Jarvinen, H. Liver fat is increased in type 2 diabetic patients and underestimated by serum alanine aminotransferase compared with equally obese nondiabetic subjects. Diabetes Care 2008, 31, 165–169. [Google Scholar] [CrossRef]
- Gastaldelli, A.; Cusi, K.; Pettiti, M.; Hardies, J.; Miyazaki, Y.; Berria, R.; Buzzigoli, E.; Sironi, A.M.; Cersosimo, E.; Ferrannini, E.; et al. Relationship between hepatic/visceral fat and hepatic insulin resistance in nondiabetic and type 2 diabetic subjects. Gastroenterology 2007, 133, 496–506. [Google Scholar] [CrossRef]
- Bellentani, S.; Saccoccio, G.; Masutti, F.; Croce, L.S.; Brandi, G.; Sasso, F.; Cristanini, G.; Tiribelli, C. Prevalence of and risk factors for hepatic steatosis in Northern Italy. Ann. Intern. Med. 2000, 132, 112–117. [Google Scholar]
- Bhatia, L.S.; Curzen, N.P.; Calder, P.C.; Byrne, C.D. Non-alcoholic fatty liver disease: a new and important cardiovascular risk factor? Eur. Heart J. 2012, 33, 1190–1200. [Google Scholar] [CrossRef]
- Sanyal, A.J.; Campbell-Sargent, C.; Mirshahi, F.; Rizzo, W.B.; Contos, M.J.; Sterling, R.K.; Luketic, V.A.; Shiffman, M.L.; Clore, J.N. Nonalcoholic steatohepatitis: Association of insulin resistance and mitochondrial abnormalities. Gastroenterology 2001, 120, 1183–1192. [Google Scholar] [CrossRef]
- Yki-Jarvinen, H. Liver fat in the pathogenesis of insulin resistance and type 2 diabetes. Dig. Dis. 2010, 28, 203–209. [Google Scholar] [CrossRef]
- Fabbrini, E.; Magkos, F.; Mohammed, B.S.; Pietka, T.; Abumrad, N.A.; Patterson, B.W.; Okunade, A.; Klein, S. Intrahepatic fat, not visceral fat, is linked with metabolic complications of obesity. Proc. Natl. Acad. Sci. USA 2009, 106, 15430–15435. [Google Scholar] [CrossRef]
- Bugianesi, E.; Gastaldelli, A.; Vanni, E.; Gambino, R.; Cassader, M.; Baldi, S.; Ponti, V.; Pagano, G.; Ferrannini, E.; Rizzetto, M. Insulin resistance in non-diabetic patients with non-alcoholic fatty liver disease: Sites and mechanisms. Diabetologia 2005, 48, 634–642. [Google Scholar] [CrossRef]
- Kantartzis, K.; Peter, A.; Machicao, F.; Machann, J.; Wagner, S.; Konigsrainer, I.; Konigsrainer, A.; Schick, F.; Fritsche, A.; Haring, H.U.; et al. Dissociation between fatty liver and insulin resistance in humans carrying a variant of the patatin-like phospholipase 3 gene. Diabetes 2009, 58, 2616–2623. [Google Scholar] [CrossRef]
- Sevastianova, K.; Kotronen, A.; Gastaldelli, A.; Perttila, J.; Hakkarainen, A.; Lundbom, J.; Suojanen, L.; Orho-Melander, M.; Lundbom, N.; Ferrannini, E.; et al. Genetic variation in PNPLA3 (adiponutrin) confers sensitivity to weight loss-induced decrease in liver fat in humans. Am. J. Clin. Nutr. 2011, 94, 104–111. [Google Scholar] [CrossRef]
- Amaro, A.; Fabbrini, E.; Kars, M.; Yue, P.; Schechtman, K.; Schonfeld, G.; Klein, S. Dissociation between intrahepatic triglyceride content and insulin resistance in familial hypobetalipoproteinemia. Gastroenterology 2010, 139, 149–153. [Google Scholar] [CrossRef]
- Visser, M.E.; Lammers, N.M.; Nederveen, A.J.; van der Graaf, M.; Heerschap, A.; Ackermans, M.T.; Sauerwein, H.P.; Stroes, E.S.; Serlie, M.J. Hepatic steatosis does not cause insulin resistance in people with familial hypobetalipoproteinaemia. Diabetologia 2011, 54, 2113–2121. [Google Scholar] [CrossRef]
- Kantartzis, K.; Machicao, F.; Machann, J.; Schick, F.; Fritsche, A.; Haring, H.U.; Stefan, N. The DGAT2 gene is a candidate for the dissociation between fatty liver and insulin resistance in humans. Clin. Sci. (Lond.) 2009, 116, 531–537. [Google Scholar] [CrossRef]
- Monetti, M.; Levin, M.C.; Watt, M.J.; Sajan, M.P.; Marmor, S.; Hubbard, B.K.; Stevens, R.D.; Bain, J.R.; Newgard, C.B.; Farese, R.V., Sr.; et al. Dissociation of hepatic steatosis and insulin resistance in mice overexpressing DGAT in the liver. Cell Metab. 2007, 6, 69–78. [Google Scholar] [CrossRef]
- Targher, G.; Bertolini, L.; Padovani, R.; Rodella, S.; Tessari, R.; Zenari, L.; Day, C.; Arcaro, G. Prevalence of nonalcoholic fatty liver disease and its association with cardiovascular disease among type 2 diabetic patients. Diabetes Care 2007, 30, 1212–1218. [Google Scholar] [CrossRef]
- Lomonaco, R.; Ortiz-Lopez, C.; Orsak, B.; Webb, A.; Hardies, J.; Darland, C.; Finch, J.; Gastaldelli, A.; Harrison, S.; Tio, F.; et al. Effect of adipose tissue insulin resistance on metabolic parameters and liver histology in obese patients with nonalcoholic fatty liver disease. Hepatology 2012, 55, 1389–1397. [Google Scholar] [CrossRef]
- Arner, P. Insulin resistance in type 2 diabetes: Role of fatty acids. Diabetes Metab. Res. Rev. 2002, 18 (Suppl. 2), S5–S9. [Google Scholar] [CrossRef]
- Korenblat, K.M.; Fabbrini, E.; Mohammed, B.S.; Klein, S. Liver, muscle, and adipose tissue insulin action is directly related to intrahepatic triglyceride content in obese subjects. Gastroenterology 2008, 134, 1369–1375. [Google Scholar] [CrossRef]
- Gastaldelli, A.; Harrison, S.A.; Belfort-Aguilar, R.; Hardies, L.J.; Balas, B.; Schenker, S.; Cusi, K. Importance of changes in adipose tissue insulin resistance to histological response during thiazolidinedione treatment of patients with nonalcoholic steatohepatitis. Hepatology 2009, 50, 1087–1093. [Google Scholar] [CrossRef]
- Fabbrini, E.; Mohammed, B.S.; Magkos, F.; Korenblat, K.M.; Patterson, B.W.; Klein, S. Alterations in adipose tissue and hepatic lipid kinetics in obese men and women with nonalcoholic fatty liver disease. Gastroenterology 2008, 134, 424–431. [Google Scholar]
- Adiels, M.; Taskinen, M.R.; Packard, C.; Caslake, M.J.; Soro-Paavonen, A.; Westerbacka, J.; Vehkavaara, S.; Hakkinen, A.; Olofsson, S.O.; Yki-Jarvinen, H.; et al. Overproduction of large VLDL particles is driven by increased liver fat content in man. Diabetologia 2006, 49, 755–765. [Google Scholar] [CrossRef]
- Miles, J.M.; Nelson, R.H. Contribution of triglyceride-rich lipoproteins to plasma free fatty acids. Horm. Metab. Res. 2007, 39, 726–729. [Google Scholar] [CrossRef]
- Machado, M.V.; Ferreira, D.M.; Castro, R.E.; Silvestre, A.R.; Evangelista, T.; Coutinho, J.; Carepa, F.; Costa, A.; Rodrigues, C.M.; Cortez-Pinto, H. Liver and muscle in morbid obesity: The interplay of fatty liver and insulin resistance. PLoS One 2012, 7, e31738. [Google Scholar] [CrossRef]
- Hwang, J.H.; Stein, D.T.; Barzilai, N.; Cui, M.H.; Tonelli, J.; Kishore, P.; Hawkins, M. Increased intrahepatic triglyceride is associated with peripheral insulin resistance: in vivo MR imaging and spectroscopy studies. Am. J. Physiol. Endocrinol. Metab. 2007, 293, E1663–E1669. [Google Scholar] [CrossRef]
- Choi, S.S.; Diehl, A.M. Hepatic triglyceride synthesis and nonalcoholic fatty liver disease. Curr. Opin. Lipidol. 2008, 19, 295–300. [Google Scholar] [CrossRef]
- Neuschwander-Tetri, B.A. Nontriglyceride hepatic lipotoxicity: the new paradigm for the pathogenesis of NASH. Curr. Gastroenterol. Rep. 2010, 12, 49–56. [Google Scholar] [CrossRef]
- Heni, M.; Machann, J.; Staiger, H.; Schwenzer, N.F.; Peter, A.; Schick, F.; Claussen, C.D.; Stefan, N.; Haring, H.U.; Fritsche, A. Pancreatic fat is negatively associated with insulin secretion in individuals with impaired fasting glucose and/or impaired glucose tolerance: A nuclear magnetic resonance study. Diabetes Metab. Res. Rev. 2010, 26, 200–205. [Google Scholar] [CrossRef]
- Kotronen, A.; Yki-Jarvinen, H.; Sevastianova, K.; Bergholm, R.; Hakkarainen, A.; Pietilainen, K.H.; Juurinen, L.; Lundbom, N.; Sorensen, T.I. Comparison of the relative contributions of intra-abdominal and liver fat to components of the metabolic syndrome. Obesity (Silver Spring) 2011, 19, 23–28. [Google Scholar] [CrossRef]
- Wald, D.; Teucher, B.; Dinkel, J.; Kaaks, R.; Delorme, S.; Boeing, H.; Seidensaal, K.; Meinzer, H.P.; Heimann, T. Automatic quantification of subcutaneous and visceral adipose tissue from whole-body magnetic resonance images suitable for large cohort studies. J. Magn. Reson. Imaging 2012, 36, 1421–1434. [Google Scholar] [CrossRef]
- Kotronen, A.; Juurinen, L.; Tiikkainen, M.; Vehkavaara, S.; Yki-Jarvinen, H. Increased liver fat, impaired insulin clearance, and hepatic and adipose tissue insulin resistance in type 2 diabetes. Gastroenterology 2008, 135, 122–130. [Google Scholar] [CrossRef]
- Gastaldelli, A. Role of beta-cell dysfunction, ectopic fat accumulation and insulin resistance in the pathogenesis of type 2 diabetes mellitus. Diabetes Res. Clin. Pract. 2011, 93 (Suppl. 1), S60–S65. [Google Scholar] [CrossRef]
- Fabbrini, E.; Tamboli, R.A.; Magkos, F.; Marks-Shulman, P.A.; Eckhauser, A.W.; Richards, W.O.; Klein, S.; Abumrad, N.N. Surgical removal of omental fat does not improve insulin sensitivity and cardiovascular risk factors in obese adults. Gastroenterology 2010, 139, 448–455. [Google Scholar] [CrossRef]
- Albu, J.B.; Curi, M.; Shur, M.; Murphy, L.; Matthews, D.E.; Pi-Sunyer, F.X. Systemic resistance to the antilipolytic effect of insulin in black and white women with visceral obesity. Am. J. Physiol. 1999, 277, E551–E560. [Google Scholar]
- Wajchenberg, B.L. Subcutaneous and visceral adipose tissue: their relation to the metabolic syndrome. Endocr. Rev. 2000, 21, 697–738. [Google Scholar] [CrossRef]
- Jensen, M.D. Role of body fat distribution and the metabolic complications of obesity. J. Clin. Endocrinol. Metab. 2008, 93, S57–S63. [Google Scholar] [CrossRef]
- Moore, M.C.; Connolly, C.C.; Cherrington, A.D. Autoregulation of hepatic glucose production. Eur. J. Endocrinol. 1998, 138, 240–248. [Google Scholar] [CrossRef]
- Gastaldelli, A.; Baldi, S.; Pettiti, M.; Toschi, E.; Camastra, S.; Natali, A.; Landau, B.R.; Ferrannini, E. Influence of obesity and type 2 diabetes on gluconeogenesis and glucose output in humans: a quantitative study. Diabetes 2000, 49, 1367–1373. [Google Scholar] [CrossRef]
- Jenssen, T.; Nurjhan, N.; Consoli, A.; Gerich, J.E. Failure of substrate-induced gluconeogenesis to increase overall glucose appearance in normal humans. Demonstration of hepatic autoregulation without a change in plasma glucose concentration. J. Clin. Invest. 1990, 86, 489–497. [Google Scholar] [CrossRef]
- Gastaldelli, A.; Kozakova, M.; Hojlund, K.; Flyvbjerg, A.; Favuzzi, A.; Mitrakou, A.; Balkau, B. Fatty liver is associated with insulin resistance, risk of coronary heart disease, and early atherosclerosis in a large European population. Hepatology 2009, 49, 1537–1544. [Google Scholar] [CrossRef]
- Sunny, N.E.; Parks, E.J.; Browning, J.D.; Burgess, S.C. Excessive hepatic mitochondrial TCA cycle and gluconeogenesis in humans with nonalcoholic fatty liver disease. Cell Metab. 2011, 14, 804–810. [Google Scholar] [CrossRef]
- Gastaldelli, A.; Miyazaki, Y.; Pettiti, M.; Matsuda, M.; Mahankali, S.; Santini, E.; DeFronzo, R.A.; Ferrannini, E. Metabolic effects of visceral fat accumulation in type 2 diabetes. J. Clin. Endocrinol. Metab. 2002, 87, 5098–5103. [Google Scholar] [CrossRef]
- Boden, G.; Chen, X.; Capulong, E.; Mozzoli, M. Effects of free fatty acids on gluconeogenesis and autoregulation of glucose production in type 2 diabetes. Diabetes 2001, 50, 810–816. [Google Scholar] [CrossRef]
- Wajngot, A.; Chandramouli, V.; Schumann, W.C.; Ekberg, K.; Jones, P.K.; Efendic, S.; Landau, B.R. Quantitative contributions of gluconeogenesis to glucose production during fasting in type 2 diabetes mellitus. Metabolism 2001, 50, 47–52. [Google Scholar] [CrossRef]
- Souza, M.R.; Diniz Mde, F.; Medeiros-Filho, J.E.; Araujo, M.S. Metabolic syndrome and risk factors for non-alcoholic fatty liver disease. Arq. Gastroenterol. 2012, 49, 89–96. [Google Scholar] [CrossRef]
- Barrows, B.R.; Timlin, M.T.; Parks, E.J. Spillover of dietary fatty acids and use of serum nonesterified fatty acids for the synthesis of VLDL-triacylglycerol under two different feeding regimens. Diabetes 2005, 54, 2668–2673. [Google Scholar] [CrossRef]
- Chapman, M.J.; Ginsberg, H.N.; Amarenco, P.; Andreotti, F.; Boren, J.; Catapano, A.L.; Descamps, O.S.; Fisher, E.; Kovanen, P.T.; Kuivenhoven, J.A.; et al. Triglyceride-rich lipoproteins and high-density lipoprotein cholesterol in patients at high risk of cardiovascular disease: evidence and guidance for management. Eur. Heart J. 2011, 32, 1345–1361. [Google Scholar] [CrossRef]
- Bugianesi, E.; Pagotto, U.; Manini, R.; Vanni, E.; Gastaldelli, A.; de Iasio, R.; Gentilcore, E.; Natale, S.; Cassader, M.; Rizzetto, M.; et al. Plasma adiponectin in nonalcoholic fatty liver is related to hepatic insulin resistance and hepatic fat content, not to liver disease severity. J. Clin. Endocrinol. Metab. 2005, 90, 3498–3504. [Google Scholar] [CrossRef]
- Gastaldelli, A.; Harrison, S.; Belfort-Aguiar, R.; Hardies, J.; Balas, B.; Schenker, S.; Cusi, K. Pioglitazone in the treatment of NASH: the role of adiponectin. Aliment. Pharmacol. Ther. 2010, 32, 769–775. [Google Scholar] [CrossRef]
- Yamauchi, T.; Kamon, J.; Minokoshi, Y.; Ito, Y.; Waki, H.; Uchida, S.; Yamashita, S.; Noda, M.; Kita, S.; Ueki, K.; et al. Adiponectin stimulates glucose utilization and fatty-acid oxidation by activating AMP-activated protein kinase. Nat. Med. 2002, 8, 1288–1295. [Google Scholar] [CrossRef]
- Marchesini, G.; Brizi, M.; Bianchi, G.; Tomassetti, S.; Bugianesi, E.; Lenzi, M.; McCullough, A.J.; Natale, S.; Forlani, G.; Melchionda, N. Nonalcoholic fatty liver disease: A feature of the metabolic syndrome. Diabetes 2001, 50, 1844–1850. [Google Scholar] [CrossRef]
- Aygun, C.; Kocaman, O.; Sahin, T.; Uraz, S.; Eminler, A.T.; Celebi, A.; Senturk, O.; Hulagu, S. Evaluation of metabolic syndrome frequency and carotid artery intima-media thickness as risk factors for atherosclerosis in patients with nonalcoholic fatty liver disease. Dig. Dis. Sci. 2007, 53, 1352–1357. [Google Scholar]
- Marchesini, G.; Bugianesi, E.; Forlani, G.; Cerrelli, F.; Lenzi, M.; Manini, R.; Natale, S.; Vanni, E.; Villanova, N.; Melchionda, N.; et al. Nonalcoholic fatty liver, steatohepatitis, and the metabolic syndrome. Hepatology 2003, 37, 917–923. [Google Scholar] [CrossRef]
- Kantartzis, K.; Rittig, K.; Cegan, A.; Machann, J.; Schick, F.; Balletshofer, B.; Fritsche, A.; Schleicher, E.; Haring, H.U.; Stefan, N. Fatty liver is independently associated with alterations in circulating HDL2 and HDL3 subfractions. Diabetes Care 2008, 31, 366–368. [Google Scholar]
- Toledo, F.G.; Sniderman, A.D.; Kelley, D.E. Influence of hepatic steatosis (fatty liver) on severity and composition of dyslipidemia in type 2 diabetes. Diabetes Care 2006, 29, 1845–1850. [Google Scholar] [CrossRef]
- Sung, K.C.; Wild, S.H.; Kwag, H.J.; Byrne, C.D. Fatty liver, insulin resistance, and features of metabolic syndrome: relationships with coronary artery calcium in 10,153 people. Diabetes Care 2012, 35, 2359–2364. [Google Scholar] [CrossRef]
- Vanni, E.; Bugianesi, E.; Kotronen, A.; De Minicis, S.; Yki-Jarvinen, H.; Svegliati-Baroni, G. From the metabolic syndrome to NAFLD or vice versa? Dig. Liver Dis. 2010, 42, 320–330. [Google Scholar] [CrossRef]
- Gastaldelli, A. Fatty liver disease: the hepatic manifestation of metabolic syndrome. Hypertens. Res. 2010, 33, 546–547. [Google Scholar] [CrossRef]
- Ribeireiro, T.; Swain, J.; Sarr, M.; Kendrick, M.; Que, F.; Sanderson, S.; Krishnan, A.; Viker, K.; Watt, K.; Charlton, M. NAFLD and insulin resistance do not increase the risk of postoperative complications among patients undergoing bariatric surgery––A prospective analysis. Obes. Surg. 2011, 21, 310–315. [Google Scholar] [CrossRef]
- Huang, H.L.; Lin, W.Y.; Lee, L.T.; Wang, H.H.; Lee, W.J.; Huang, K.C. Metabolic syndrome is related to nonalcoholic steatohepatitis in severely obese subjects. Obes. Surg. 2007, 17, 1457–1463. [Google Scholar] [CrossRef]
- Sironi, A.M.; Sicari, R.; Folli, F.; Gastaldelli, A. Ectopic fat storage, insulin resistance, and hypertension. Curr. Pharm. Des. 2011, 17, 3074–3080. [Google Scholar] [CrossRef]
- Gastaldelli, A.; Basta, G. Ectopic fat and cardiovascular disease: What is the link? Nutr. Metab. Cardiovasc. Dis. 2010, 20, 481–490. [Google Scholar] [CrossRef]
- Ghouri, N.; Preiss, D.; Sattar, N. Liver enzymes, nonalcoholic fatty liver disease, and incident cardiovascular disease: A narrative review and clinical perspective of prospective data. Hepatology 2010, 52, 1156–1161. [Google Scholar] [CrossRef]
- Ortiz-Lopez, C.; Lomonaco, R.; Orsak, B.; Finch, J.; Chang, Z.; Kochunov, V.G.; Hardies, J.; Cusi, K. Prevalence of prediabetes and diabetes and metabolic profile of patients with nonalcoholic fatty liver disease (NAFLD). Diabetes Care 2012, 35, 873–878. [Google Scholar] [CrossRef]
- Targher, G.; Zoppini, G.; Day, C.P. Risk of all-cause and cardiovascular mortality in patients with chronic liver disease. Gut 2011, 60, 1602–1603; author reply 1603–1604. [Google Scholar] [CrossRef]
- Targher, G.; Bertolini, L.; Padovani, R.; Poli, F.; Scala, L.; Tessari, R.; Zenari, L.; Falezza, G. Increased prevalence of cardiovascular disease in Type 2 diabetic patients with non-alcoholic fatty liver disease. Diabet. Med. 2006, 23, 403–409. [Google Scholar] [CrossRef]
- Yki-Jarvinen, H. Fat in the liver and insulin resistance. Ann. Med. 2005, 37, 347–356. [Google Scholar] [CrossRef]
- Auberger, P.; Falquerho, L.; Contreres, J.O.; Pages, G.; Le Cam, G.; Rossi, B.; Le Cam, A. Characterization of a natural inhibitor of the insulin receptor tyrosine kinase: cDNA cloning, purification, and anti-mitogenic activity. Cell 1989, 58, 631–640. [Google Scholar] [CrossRef]
- Pal, D.; Dasgupta, S.; Kundu, R.; Maitra, S.; Das, G.; Mukhopadhyay, S.; Ray, S.; Majumdar, S.S.; Bhattacharya, S. Fetuin-A acts as an endogenous ligand of TLR4 to promote lipid-induced insulin resistance. Nat. Med. 2012, 18, 1279–1285. [Google Scholar] [CrossRef]
- Hennige, A.M.; Staiger, H.; Wicke, C.; Machicao, F.; Fritsche, A.; Haring, H.U.; Stefan, N. Fetuin-A induces cytokine expression and suppresses adiponectin production. PLoS One 2008, 3, e1765. [Google Scholar] [CrossRef]
- Dogru, T.; Genc, H.; Tapan, S.; Aslan, F.; Ercin, C.N.; Ors, F.; Kara, M.; Sertoglu, E.; Karslioglu, Y.; Bagci, S.; et al. Plasma fetuin-A is associated with endothelial dysfunction and subclinical atherosclerosis in subjects with nonalcoholic fatty liver disease. Clin. Endocrinol. (Oxf.) 2013, 78, 712–717. [Google Scholar] [CrossRef]
- Rittig, K.; Thamer, C.; Haupt, A.; Machann, J.; Peter, A.; Balletshofer, B.; Fritsche, A.; Haring, H.U.; Stefan, N. High plasma fetuin-A is associated with increased carotid intima-media thickness in a middle-aged population. Atherosclerosis 2009, 207, 341–342. [Google Scholar] [CrossRef]
- Weikert, C.; Stefan, N.; Schulze, M.B.; Pischon, T.; Berger, K.; Joost, H.G.; Haring, H.U.; Boeing, H.; Fritsche, A. Plasma fetuin-a levels and the risk of myocardial infarction and ischemic stroke. Circulation 2008, 118, 2555–2562. [Google Scholar] [CrossRef]
- Stefan, N.; Fritsche, A.; Weikert, C.; Boeing, H.; Joost, H.G.; Haring, H.U.; Schulze, M.B. Plasma fetuin-A levels and the risk of type 2 diabetes. Diabetes 2008, 57, 2762–2767. [Google Scholar] [CrossRef]
- Villanova, N.; Moscatiello, S.; Ramilli, S.; Bugianesi, E.; Magalotti, D.; Vanni, E.; Zoli, M.; Marchesini, G. Endothelial dysfunction and cardiovascular risk profile in nonalcoholic fatty liver disease. Hepatology 2005, 42, 473–480. [Google Scholar] [CrossRef]
- Kozakova, M.; Palombo, C.; Eng, M.P.; Dekker, J.; Flyvbjerg, A.; Mitrakou, A.; Gastaldelli, A.; Ferrannini, E. Fatty liver index, gamma-glutamyltransferase, and early carotid plaques. Hepatology 2012, 55, 1406–1415. [Google Scholar] [CrossRef]
- Bonapace, S.; Perseghin, G.; Molon, G.; Canali, G.; Bertolini, L.; Zoppini, G.; Barbieri, E.; Targher, G. Nonalcoholic fatty liver disease is associated with left ventricular diastolic dysfunction in patients with type 2 diabetes. Diabetes Care 2012, 35, 389–395. [Google Scholar] [CrossRef]
- Mofrad, P.; Contos, M.J.; Haque, M.; Sargeant, C.; Fisher, R.A.; Luketic, V.A.; Sterling, R.K.; Shiffman, M.L.; Stravitz, R.T.; Sanyal, A.J. Clinical and histologic spectrum of nonalcoholic fatty liver disease associated with normal ALT values. Hepatology 2003, 37, 1286–1292. [Google Scholar] [CrossRef]
- Ricci, C.; Longo, R.; Gioulis, E.; Bosco, M.; Pollesello, P.; Masutti, F.; Croce, L.S.; Paoletti, S.; de Bernard, B.; Tiribelli, C.; et al. Noninvasive in vivo quantitative assessment of fat content in human liver. J. Hepatol. 1997, 27, 108–113. [Google Scholar] [CrossRef]
- Tsuneto, A.; Hida, A.; Sera, N.; Imaizumi, M.; Ichimaru, S.; Nakashima, E.; Seto, S.; Maemura, K.; Akahoshi, M. Fatty liver incidence and predictive variables. Hypertens. Res. 2010, 33, 638–643. [Google Scholar] [CrossRef]
- Bedogni, G.; Bellentani, S.; Miglioli, L.; Masutti, F.; Passalacqua, M.; Castiglione, A.; Tiribelli, C. The Fatty Liver Index: A simple and accurate predictor of hepatic steatosis in the general population. BMC Gastroenterol. 2006, 6, 33. [Google Scholar] [CrossRef]
- Targher, G.; Day, C.P.; Bonora, E. Risk of cardiovascular disease in patients with nonalcoholic fatty liver disease. N. Engl. J. Med. 2010, 363, 1341–1350. [Google Scholar] [CrossRef]
- Hill, J.M.; Zalos, G.; Halcox, J.P.; Schenke, W.H.; Waclawiw, M.A.; Quyyumi, A.A.; Finkel, T. Circulating endothelial progenitor cells, vascular function, and cardiovascular risk. N. Engl. J. Med. 2003, 348, 593–600. [Google Scholar] [CrossRef]
- Chiang, C.H.; Huang, P.H.; Chung, F.P.; Chen, Z.Y.; Leu, H.B.; Huang, C.C.; Wu, T.C.; Chen, J.W.; Lin, S.J. Decreased circulating endothelial progenitor cell levels and function in patients with nonalcoholic fatty liver disease. PLoS One 2012, 7, e31799. [Google Scholar] [CrossRef]
- Lin, Y.C.; Lo, H.M.; Chen, J.D. Sonographic fatty liver, overweight and ischemic heart disease. World J. Gastroenterol. 2005, 11, 4838–4842. [Google Scholar]
- Bugianesi, E.; Gastaldelli, A. Hepatic and cardiac steatosis: Are they coupled? Heart Fail. Clin. 2012, 8, 663–670. [Google Scholar] [CrossRef]
- Assy, N.; Djibre, A.; Farah, R.; Grosovski, M.; Marmor, A. Presence of coronary plaques in patients with nonalcoholic fatty liver disease. Radiology 2010, 254, 393–400. [Google Scholar] [CrossRef]
- Ebrahimi, M.; Kazemi-Bajestani, S.M.; Ghayour-Mobarhan, M.; Moohebati, M.; Paydar, R.; Azimi-Nezhad, M.; Esmaily, H.O.; Ferns, G.A. Metabolic syndrome may not be a good predictor of coronary artery disease in the Iranian population: population-specific definitions are required. Sci. World J. 2009, 9, 86–96. [Google Scholar] [CrossRef]
© 2013 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).