SEARCH

SEARCH BY CITATION

Keywords:

  • insulin resistance;
  • non-alcoholic steatohepatitis

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Box 1
  5. Development of NAFLD
  6. Dietary lipid and carbohydrate and NAFLD
  7. How is NAFLD diagnosed?
  8. NAFLD and its association with Type 2 diabetes and cardiovascular disease
  9. Potential treatments for NAFLD
  10. Box 2
  11. Conclusions
  12. Competing interests
  13. Acknowledgements
  14. References
  15. Supporting Information

Diabet. Med. 29, 1098–1107 (2012)

Abstract

Non-alcoholic fatty liver disease is now recognized as the hepatic component of the metabolic syndrome. Non-alcoholic fatty liver disease is a spectrum of fat-associated liver conditions that can result in end-stage liver disease and the need for liver transplantation. Simple steatosis, or fatty liver, occurs early in non-alcoholic fatty liver disease and may progress to non-alcoholic steatohepatitis, fibrosis and cirrhosis with increased risk of hepatocellular carcinoma. Prevalence estimates for non-alcoholic fatty liver disease range from 17 to 33% in the general populations and it has been estimated that non-alcoholic fatty liver disease exists in up to 70% of people with Type 2 diabetes. Non-alcoholic fatty liver disease increases risk of Type 2 diabetes and cardiovascular disease. In people with Type 2 diabetes, non-alcoholic fatty liver disease is the most frequent cause (∼80%) of fatty liver diagnosed by ultrasound. As non-alcoholic fatty liver disease is strongly associated with insulin resistance, the presence of non-alcoholic fatty liver disease with diabetes often contributes to poor glycaemic control. Consequently, strategies that decrease liver fat and improve whole-body insulin sensitivity may both contribute to prevention of Type 2 diabetes and to better glycaemic control in people who already have developed diabetes. This review summarizes the Dorothy Hodgkin lecture given by the author at the 2012 Diabetes UK annual scientific conference, proposing that fatty acid fluxes through the liver are crucial for the pathogenesis of non-alcoholic fatty liver disease and for increasing insulin resistance.


Abbreviations
NAFLD

non-alcoholic fatty liver disease

NASH

non-alcoholic steatohepatitis

Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Box 1
  5. Development of NAFLD
  6. Dietary lipid and carbohydrate and NAFLD
  7. How is NAFLD diagnosed?
  8. NAFLD and its association with Type 2 diabetes and cardiovascular disease
  9. Potential treatments for NAFLD
  10. Box 2
  11. Conclusions
  12. Competing interests
  13. Acknowledgements
  14. References
  15. Supporting Information

The use of the term metabolic syndrome places a focus on central or ‘ectopic’ fat accumulation and linked cardio-metabolic risk factors. Ectopic fat accumulation in tissues such as liver is termed non-alcoholic fatty liver disease (NAFLD) and it is now evident that this condition is potentially harmful and associated with important liver and cardio-metabolic consequences [1–3].

NAFLD has become one of the most common causes of chronic liver disease worldwide, causing considerable liver morbidity and mortality, and is now becoming a major reason for liver transplantation in order to rescue patients with end-stage liver disease. Often patients are asymptomatic and are diagnosed as part of investigation for incidental abnormal liver function tests. A typical presentation of a patient with NAFLD is shown in Box 1 and the diagnostic tests that are used to diagnose NAFLD are discussed below.

It is now clear that NAFLD is also a risk factor for Type 2 diabetes and cardiovascular disease [4], but the mechanisms by which NAFLD [5] causes both diseases are unclear and need to be better elucidated [3,6]. Like many chronic disorders, NAFLD is not a single disease entity, but the term NAFLD describes a spectrum of liver conditions. The spectrum of disorders ranges from simple fatty liver (steatosis) to more severe steatosis coupled with marked inflammation, termed non-alcoholic steatohepatitis (NASH). Importantly, NASH is often progressive with development of fibrosis (40–50%), liver cirrhosis (15–17%), liver failure (3%) and, potentially, hepatocellular carcinoma. Current estimates are that 40% of people with NAFLD develop NASH [7], and there is increased incidence of coronary (10.8%), cerebrovascular (37.3%) and peripheral (24.5%) vascular disease in individuals with NAFLD [8]. Consequently, with no licensed medication available for NAFLD, new treatments are urgently needed.

As NAFLD is strongly associated with insulin resistance, which occurs in other tissues besides liver [9], the presence of NAFLD in people with Type 2 diabetes often makes it difficult to obtain good glycaemic control. Consequently, strategies to decrease liver fat per se and improving whole-body insulin sensitivity may both contribute to prevention of Type 2 diabetes and to better glycaemic control in people with diabetes.

Box 1

  1. Top of page
  2. Abstract
  3. Introduction
  4. Box 1
  5. Development of NAFLD
  6. Dietary lipid and carbohydrate and NAFLD
  7. How is NAFLD diagnosed?
  8. NAFLD and its association with Type 2 diabetes and cardiovascular disease
  9. Potential treatments for NAFLD
  10. Box 2
  11. Conclusions
  12. Competing interests
  13. Acknowledgements
  14. References
  15. Supporting Information

Case report

  •  45-year-old man
  • o
    Asymptomatic
  • o
    Smokes 10 cigarettes/day—20 years
  • o
    Type 2 diabetes—1 year
  • o
    Sedentary occupation, 14–21 units alcohol/week
  • o
    Married, two children
  • o
    ‘Unfit and stressed at work’
  • o
    Metformin 2 g/day

On examination

  •  BMI 36 kg/m2
  •  ‘Increased waist circumference’ 104 cms
  •  Blood pressure 145/90 mmHg
  • o
    Fasting glucose = 9.0 mmol/l
  • o
    Cholesterol 6.0 mmol/l, LDL = 3.2 mmol/l
  • o
    Fasting triglyceride = 4.0 mmol/l
  • o
    HDL cholesterol = 0.8 mmol/l
  • o
    HbA1c = 75 mmol/mol (9.0%)
  •  Note all five features of the metabolic syndrome
  •  ALT = 50 iu/l (5–35 iu/l)
  •  GGT = 50 iu/l (< 55 iu/l)
  •  Liver function tests otherwise entirely normal
  •  Urea and electrolytes normal
  •  Urinalysis—trace protein

Albumin:creatinine ratio = 2.5 mg/mmol

Investigations

Ultrasound

  •  ‘Echogenic liver texture in keeping with moderate to severe fatty liver’
  •  Serum hyaluronic acid normal and procollagen III amino terminal peptide (PIIINP) concentrations increased

Note: exclude other causes of fatty liver

  • o
    Alcohol intake—minimal
  • o
    Iron status—normal
  • o
    Autoantibodies—slight increase anti-smooth cell immunoglobulin G
  • o
    Immunoglobulins—normal
  • o
    Hepatitis status (Hep C infection) negative

Development of NAFLD

  1. Top of page
  2. Abstract
  3. Introduction
  4. Box 1
  5. Development of NAFLD
  6. Dietary lipid and carbohydrate and NAFLD
  7. How is NAFLD diagnosed?
  8. NAFLD and its association with Type 2 diabetes and cardiovascular disease
  9. Potential treatments for NAFLD
  10. Box 2
  11. Conclusions
  12. Competing interests
  13. Acknowledgements
  14. References
  15. Supporting Information

Is fatty acid flux through the liver key to the pathogenesis of NAFLD?

Fat accumulation in the liver is influenced by the delivery of dietary fat to the liver (contribution to liver fat ∼5%); delivery of extra-hepatic non-esterified fatty acids (NEFAs) to the liver (contribution to liver fat ∼60%); and the remainder of liver fat accumulation is affected by hepatic de novo lipogenesis [2]. Fatty acids from adipose tissue lipolysis, from dietary chylomicrons and arising from de novo lipogenesis are partitioned within the liver into different pools with different fates [e.g. secretion as lipoprotein (VLDL), oxidation, bile acid formation, phospholipid assembly] (Fig. 1). The fluxes of these fatty acids through the different pools can be measured with substrate labelling techniques (for review, see Hodson and Frayn [10]). Plasma fatty acid concentrations are increased in people with fatty liver [9] and in people with slight abnormalities of glucose tolerance [11] and Type 2 diabetes [12], and increased fatty acid flux both to the liver and through the liver may contribute to the development of liver fat accumulation and hepatic inflammation. With development of the more severe forms of NAFLD, such as NASH with fibrosis, there is increased insulin resistance, potentially setting up a vicious cycle of insulin resistance, increased fatty acid supply to the liver and increased hepatic steatosis and liver inflammation (as illustrated in Fig. 2). We have tested the effect of increasing the fatty acid supply to the developing fetal liver and addressed the question of whether this exposure affects the predisposition to NAFLD in the offspring later in adult life. These experiments undertaken in mice showed that increasing the content of dietary fat in the mother’s diet during pregnancy increased the severity of NAFLD in the adult offspring borne of these mothers, producing a histological appearance in keeping with human NASH [13,14]. In this mouse model, we showed that mitochondrial function was impaired in liver in the offspring exposed to the altered maternal diet [14], suggesting that oxidative metabolism may be impaired in these animals. However, to date, the mechanism underpinning impaired mitochondrial function is uncertain. The links between obesity and insulin resistance that increases fatty acid flux to the liver, and the hepatic mechanisms affected by altered fatty acid fluxes that contribute to the pathogenesis of end-stage liver disease in NAFLD are shown in Fig. 3.

image

Figure 1.  Hepatic partitioning of fatty acids and regulation of fatty acid supply to the liver by insulin. CE, cholesterol ester; CO2, carbon dioxide; CoA, coenzyme A; CPT-1, carnitine palmitoyltransferase I; DAG, diacylglycerol; FA, fatty acid; PL, phospholipids; TAG, triacylglycerol (with thanks to Dr Leanne Hodson, University of Oxford, UK).

Download figure to PowerPoint

image

Figure 2.  Potential hepatic mechanisms involved in the progression of liver disease from simple steatosis to end-stage liver disease in non-alcoholic fatty liver disease (NAFLD). NASH, non-alcoholic steatohepatitis.

Download figure to PowerPoint

image

Figure 3.  Vicious cycle of worsening insulin resistance in non-alcoholic fatty liver disease (NAFLD). Initial insulin resistance and fatty acid release from adipocytes promotes hepatic steatosis. Increased fatty acid fluxes through the liver promote hepatic gluconeogenesis, worsening hepatic insulin resistance and potentially worsening of whole-body insulin resistance with adverse changes in cardio-metabolic risk factors. The net effect of these changes is: (1) increased risk of progression of liver disease; (2) increased risk of Type 2 diabetes and cardiovascular disease; and (3) increased risk of poor glycaemic control in people with established diabetes. ER, endoplasmic reticulum; FFAs, free fatty acids; IκκB, inhibitor of nuclear factor kappa-B kinase subunit beta; IL-6, interleukin 6; NEFAs, non esterified fatty acids; NFκB, nuclear factor kappa B; TAG, triacylglycerol; TNFα, tumour necrosis factor α.

Download figure to PowerPoint

Dietary lipid and carbohydrate and NAFLD

  1. Top of page
  2. Abstract
  3. Introduction
  4. Box 1
  5. Development of NAFLD
  6. Dietary lipid and carbohydrate and NAFLD
  7. How is NAFLD diagnosed?
  8. NAFLD and its association with Type 2 diabetes and cardiovascular disease
  9. Potential treatments for NAFLD
  10. Box 2
  11. Conclusions
  12. Competing interests
  13. Acknowledgements
  14. References
  15. Supporting Information

The role of dietary cholesterol, different carbohydrates and different fatty acids in the aetiology of NAFLD is poorly understood [1]. Diets high in saturated fats and cholesterol have been demonstrated to induce weight gain, insulin resistance and hyperlipidaemia in humans and animals. High dietary fat intake has been shown to induce perturbations in insulin signalling and rates of lipid synthesis via increased hepatic fatty acid flux and triglyceride that may be relevant to liver fat accumulation in NAFLD. Whilst diets high in carbohydrates, particularly fructose, have been shown to contribute to weight gain, hyperlipidaemia and metabolic disturbances, fructose may also have a specific deleterious effect on hepatic fat accumulation. It has been shown that feeding fructose for a 2-week period induces hepatic and whole-body insulin resistance, accompanied by increases in plasma triglyceride, cholesterol and non-esterified fatty acid [15]. Fructose-induced insulin resistance was associated with a considerable rise in the production of VLDL. Thus, the fructose-fed hamster may be relevant to the development of NAFLD in humans.

How is NAFLD diagnosed?

  1. Top of page
  2. Abstract
  3. Introduction
  4. Box 1
  5. Development of NAFLD
  6. Dietary lipid and carbohydrate and NAFLD
  7. How is NAFLD diagnosed?
  8. NAFLD and its association with Type 2 diabetes and cardiovascular disease
  9. Potential treatments for NAFLD
  10. Box 2
  11. Conclusions
  12. Competing interests
  13. Acknowledgements
  14. References
  15. Supporting Information

At present, a liver biopsy remains the only reliable way to diagnose NAFLD and to establish the presence of fibrosis. When liver biopsy is undertaken, the 12-point Kleiner scoring system [16] can be very useful in evaluating the severity of the liver disease within the spectrum that encapsulates NAFLD. The Kleiner score assigns up to 3 points to indicate the severity of the fat accumulation, up to 2 points for ballooning of hepatocytes (that occurs with NASH), up to 3 points for lobular inflammation and up to 4 points for the severity of the fibrosis. However, despite this benefit of knowing how severe the liver disease is, NAFLD can be a patchy disease and therefore the biopsy result can be misleading. Therefore, the sampling variability has the potential to alter significantly the diagnosis and staging of NAFLD. It is not practical to offer liver biopsy as a test for the diagnosis of NAFLD to all patients, as the prevalence of the condition would overwhelm service provision. Consequently non-invasive markers of NAFLD are urgently needed for the following reasons:

  • 1
     Diagnosis and monitoring responses to therapy. It is important to realize that many studies investigating the aetiology and pathogenesis of NAFLD, and investigating potential treatments for NAFLD, will not be able to use liver biopsy and will need to use proxy markers for all components of the disease process.
  • 2
     To establish which risk factors are aetiologically linked to the different components of the liver disease and to establish which treatments improve liver fat, liver inflammation or liver fibrosis within the spectrum of NAFLD.
  • 3
     To differentiate between patients with simple hepatic steatosis and those with NASH and liver fibrosis. A variety of different markers have been tested and these have been reviewed by Guha and colleagues [17]. Biochemical markers measured in serum, such as hyaluronic acid, tissue inhibitor of matrix metalloproteinase 1, laminin, type IV collagen and the amino terminal peptide of procollagen III (PIIINP), are increased with liver fibrosis. The European Liver Fibrosis score is derived using a proprietary algorithm that utilizes tissue inhibitor of matrix metalloproteinase 1, procollagen III and hyaluronic acid and the European Liver Fibrosis score has been tested in a small subgroup of patients with NAFLD recruited with liver fibrosis [18]. The European Liver Fibrosis score shows promise with good sensitivity and specificity for NASH with fibrosis, although further research is needed to assess its utility in people with different components of the disease process within NAFLD. Another simpler algorithm has recently been developed and published in full, utilizing simple anthropometric measurements and biochemical tests [19]. This test is also showing considerable promise with good sensitivity and specificity for NASH with fibrosis.

The challenge remains to establish biomarkers that are simple, reproducible and inexpensive, and that have high sensitivity and specificity for NAFLD, NASH and NASH with fibrosis in order to delineate the severity of NAFLD within individual patients. As diabetes is associated with the more severe forms of NAFLD, and the condition has often progressed silently to liver fibrosis, this is particularly important when coexisting diabetes is present. Identification of simple, inexpensive biomarkers would facilitate achieving reliable estimates of prevalence of NAFLD worldwide in people with diabetes and would provide a diagnostic tool for the monitoring of responses to therapeutic interventions.

Fatty liver is associated with increased serum alanine aminotransferase (ALT) and γ-glutamyltransferase (GGT) concentrations and these liver enzymes are generally considered as surrogate markers of parenchymal cell and bile duct canaliculi dysfunction. The increase in liver enzymes is mild and restricted to one or both of ALT and aspartate aminotransferase (AST) (the degree of elevation is less than that observed with alcoholic liver cirrhosis). The ratio of AST to ALT is usually less than 1 and, importantly, liver-enzyme concentrations do not correlate with histology. GGT may also be increased in NAFLD, although the increase is also seen in patients who have excess alcohol intake.

Ultrasound, computed tomography (CT) scanning, magnetic resonance spectroscopy (MRS) and magnetic resonance imaging (MRI) with in- and out-of-phase imaging have all been used in the diagnosis of NAFLD. Ultrasound has a sensitivity of 89% and specificity of 77% and is commonly used in a clinical practice setting. Quantitative assessment of fatty infiltration is best achieved with MRS and in- and out-of-phase MRI.

NAFLD and its association with Type 2 diabetes and cardiovascular disease

  1. Top of page
  2. Abstract
  3. Introduction
  4. Box 1
  5. Development of NAFLD
  6. Dietary lipid and carbohydrate and NAFLD
  7. How is NAFLD diagnosed?
  8. NAFLD and its association with Type 2 diabetes and cardiovascular disease
  9. Potential treatments for NAFLD
  10. Box 2
  11. Conclusions
  12. Competing interests
  13. Acknowledgements
  14. References
  15. Supporting Information

There is now general agreement that NAFLD is another clinical feature of the metabolic syndrome [20–23]. A study of 66 patients with NAFLD showed that 98% were insulin resistant and 39% of those subjects had diabetes [24]. In another study of 19 patients with NAFLD who were not obese or did not have diabetes and had a normal lipid profile, 47% showed features of the metabolic syndrome [25]. Obesity is associated with a higher incidence of a wide spectrum of liver diseases associated with NAFLD, from steatosis to fibrosis and cirrhosis [26]. NAFLD increases the risk of Type 2 diabetes and we have recently shown that this effect is independent of insulin resistance and overweight and obesity [27]. After adjustment for multiple potential confounders, coexisting fatty liver, insulin resistance and overweight or obesity markedly increased the odds ratio for incident diabetes (Table 1), suggesting that NAFLD, insulin resistance and overweight/obesity may each contribute to increasing the risk of Type 2 diabetes via different mechanisms.

Table 1.   Odds ratio for incident diabetes at 5-year follow-up for different combinations of insulin resistance, overweight/obesity and fatty liver at baseline
 Numbers and proportions with incident diabetes (%)Odds ratio (95% CI) Model
  1. Model adjusted for age, sex, alcohol, smoking status, exercise, educational status, triglyceride and ALT. Insulin resistance defined according to homestasis model assessment of insulin resistance > 75th centile (i.e. HOMA > 2.0); fatty liver defined by the presence of liver fat detected by ultrasound and overweight/obesity defined by BMI ≥ 25 kg/m2 (adapted from Sung et al. [27]).

Whole cohort223/12 853 (1.7%) 
No risk factors26/6324 (0.4%)1
Insulin resistance alone14/945 (1.5%)3.66 (1.89–7.08), P < 0.001
Overweight/obesity alone10/1434 (0.7%)1.29 (0.62–2.71), P = 0.50
Fatty liver alone13/850 (1.5%)2.73 (1.38–5.41), P = 0.004
Insulin resistance and overweight/obesity21/595 (3.5%)6.16 (3.38–11.22), P < 0.001
Insulin resistance and fatty liver15/388 (3.9%)6.73 (3.49–12.97), P < 0.001
Overweight/obesity and fatty liver20/1032 (1.9%)3.23 (1.78–5.89), P < 0.001
Insulin resistance, overweight/obesity and fatty/liver104/1285 (8.1%)14.13 (8.99–22.2), P < 0.001

A recent study undertaken in 19 European centres showed a strong association between reduced insulin sensitivity and increased incidence of cardiovascular disease with NAFLD [28], suggesting that it may be worth screening patients with NAFLD for diabetes. Several prospective studies have reported an increased incidence of cardiovascular events in people with NAFLD [7,29–39]. However, it is still unclear whether NAFLD is simply a risk marker that coexists in people at increased cardiovascular risk or is an independent cardiovascular risk factor. If it is proven that NAFLD is an independent risk factor for cardiovascular disease, a diagnosis of NAFLD could be used to identify subgroups of individuals for specific advice on intensive lifestyle modification that is known to be effective at deceasing fatty liver and for pharmacological treatment to decrease cardiovascular disease risk [2,40].

It has been suggested that the relationship between NAFLD and cardiovascular disease is weak [41] and, given that NAFLD, central obesity and other features of the metabolic syndrome, including diabetes, often coexist [2], it is plausible that any relationship between NAFLD and cardiovascular disease may be mediated, at least in part, by the presence of coexisting central obesity or diabetes. A recent study of the natural history of patients with NAFLD and severe fibrosis (or cirrhosis) confirms that this group of patients are at high risk of cardiovascular events [42] and there is also now evidence to suggest that people with NASH are at greater risk of cardiovascular disease than people with simple steatosis or fatty liver [7,43]. As NASH and NASH with fibrosis are more common in people with Type 2 diabetes than among people without diabetes, further work is needed to confirm that NAFLD is an independent cardiovascular disease risk factor rather than an epiphenomenon occurring in people at risk of cardiovascular disease [44].

A diagnosis of NAFLD is associated with increased risk of overall death in both individuals with diabetes and those without diabetes, and cardiovascular disease is one of the leading causes of death in this group of patients. In a study of biopsy-proven NAFLD with follow-up for approximately 21 years, the main causes of death in patients with NAFLD were cardiovascular disease and malignancy [45]. Interestingly, the histological severity of NAFLD and inflammation is strongly associated with increased risk of cardiovascular disease and an atherogenic lipid profile [46]. Simple hepatic steatosis is also associated with silent carotid atherosclerosis [47], suggesting that NAFLD may also be associated with increased risk of cerebrovascular disease.

Prospective studies have reported associations between increased liver enzymes (particularly serum GGT level) as surrogate markers of NAFLD [48,49] and the occurrence of cardiovascular disease events in both subjects without diabetes and patients with Type 2 diabetes [50,51]. Increased ALT concentrations (as a proxy for NAFLD) may be linked to an increase in risk of cardiovascular disease, independently of traditional risk factors and the features of the metabolic syndrome [52], suggesting that NAFLD is associated with cardiovascular disease independently of other features of the metabolic syndrome. Recently, the Firenze Bagno a Ripoli (FIBAR) study concluded that an increased GGT or AST were independent predictors of cardiovascular disease and an increase of GGT level above the reference range, or also in the upper reference range, was an independent predictor of incident diabetes [53].

In recent work from Targher et al. [54], the authors showed that the prevalence of NAFLD in people with diabetes was 69.5% among participants and NAFLD was the most common cause (81.5%) of hepatic steatosis detected by ultrasound. The prevalence of NAFLD increased with age and the age-adjusted prevalence of NAFLD was 71.1% in men and 68% in women in people with diabetes. Patients with NAFLD had a higher age- and sex-adjusted prevalence of coronary, cerebrovascular and peripheral vascular disease than their counterparts without NAFLD. NAFLD was associated with prevalent cardiovascular disease independent of classical risk factors, glycaemic control, medications and the metabolic syndrome features [54]. The Valpolicella Heart Diabetes Study conducted in 2103 patients with Type 2 diabetes [55] also demonstrated that NAFLD is associated with an increased risk of future cardiovascular disease events and, importantly, this association was independent of classical risk factors, liver enzymes and the metabolic syndrome. Thus, the evidence suggests that NAFLD is an independent risk factor for coronary heart disease, and possibly cerebrovascular disease and peripheral vascular disease, even after adjustment for features of the metabolic syndrome.

Potential treatments for NAFLD

  1. Top of page
  2. Abstract
  3. Introduction
  4. Box 1
  5. Development of NAFLD
  6. Dietary lipid and carbohydrate and NAFLD
  7. How is NAFLD diagnosed?
  8. NAFLD and its association with Type 2 diabetes and cardiovascular disease
  9. Potential treatments for NAFLD
  10. Box 2
  11. Conclusions
  12. Competing interests
  13. Acknowledgements
  14. References
  15. Supporting Information

To date there are no licensed treatments for NAFLD. However, several drugs licensed for other indications are now being tested in NAFLD. We have recently summarized the evidence for the efficacy of these drugs in NAFLD [3]. Although most of the focus on treatments in NAFLD has targeted liver lipid, and specifically liver triglyceride, the liver also plays a crucial role in the maintenance of cholesterol homeostasis and it is not certain whether modifying liver cholesterol content could produce a benefit in NAFLD. The liver is the organ that receives most of the cholesterol absorbed by the small intestine and also is the site for the excretion of cholesterol in bile. The liver actively synthesizes cholesterol and this is affected by the amount of cholesterol being delivered to it from the small intestine. It is well documented that dietary or pharmacological manipulation of the enterohepatic flux of either cholesterol or bile acids can potentially cause marked changes in the rate at which the liver synthesizes cholesterol, converts cholesterol to bile acids, incorporates cholesterol into VLDLs, esterifies and stores cholesterol, or secretes unesterified cholesterol directly into bile. Thus, it is plausible that the cholesterol absorption inhibitor (ezetimibe) could have a beneficial effect in NAFLD (see Ahmed and Byrne [56]).

N-3 long chain polyunsaturated fatty acids (or fish oil fatty acids) are potential agents to alter favourably liver lipid fluxes, and these agents also decrease inflammation. N-3 polyunsaturated fatty acids are also well known to decrease inflammation, involving decreased hepatic tumour necrosis factor α (TNF-α) production [57,58]. In both animal and human studies, fish oil feeding decreases the production of pro-inflammatory cytokines by immune cells [59] by inhibiting activation of the pro-inflammatory transcription factor nuclear factor kappa B (NFκB) and by activating the anti-inflammatory transcription factor peroxisome proliferator-activated receptor gamma (PPAR-γ). Recent studies have also identified a novel group of n-3 polyunsaturated fatty acid-derived mediators, termed E- and D-series resolvins (resolution-phase interaction products) that exert potent anti-inflammatory actions in neutrophils, macrophages, dendritic cells and T-cells [57,59].

The combination of obesity and insulin resistance increases flux of fatty acids to the liver from adipose tissue [9,60]. In individuals who develop NAFLD, the flux of fatty acids to the liver, exceeds the impaired capacity of the liver to dispose of these either by β-oxidation or secretion as VLDL, or as part of phosphatidyl choline in bile, resulting in excess fatty acid accumulation within hepatocytes as triglyceride. This provides a strong basis for attempting to modify the supply of fatty acids to the liver and to alter their flux with the effect of reducing hepatic de novo lipogenesis. A disruption in the hepatic lipid composition has been shown in patients with NAFLD [61] and a marked increase in long-chain n-6/n-3 polyunsaturated fatty acids ratio, attributable to n-3 polyunsaturated fatty acid depletion occurring in NAFLD, a situation favouring lipid synthesis over oxidation and secretion, thereby potentially leading to steatosis [61]. N-3 polyunsaturated fatty acids are also natural ligands of PPAR-α, nuclear receptors which modulate lipid metabolism in hepatocytes [62]. Indeed, low levels of circulating n-3 polyunsaturated fatty acids impair PPAR- α activity in the liver and this is associated with a higher hepatic uptake of circulating free fatty acids, a decrease of hepatocyte mitochondrial β-oxidation, a reduced synthesis of VLDL and an up-regulation of lipogenic transcription factors [e.g. sterol regulatory element binding protein-1 (SREBP-1)] [63–65]. Thus, there is a need to determine whether purified n-3 fatty acids are beneficial in NAFLD, as there is good theoretical evidence that these fatty acids could have a favourable effect on both liver lipid and inflammatory processes, both of which are relevant to the spectrum of liver conditions in NAFLD. Based on the supporting evidence presented here, we are presently conducting a randomized controlled trial with high-dose purified n-3 polyunsaturated fatty acids in people with NAFLD. The study is a phase-IV trial in patients with NAFLD that is testing the effects of high-dose purified n-3 long-chain fatty acids [Omacor-Solvay/Abbott/Pronova (Abbott Healthcare Products Ltd, Berkshire, UK) 4 g once daily] on a range of liver and cardio-metabolic outcomes. The trial will be completed in 2012. [The WELCOME study (Wessex Evaluation of fatty Liver and Cardiovascular markers in NAFLD (non-alcoholic fatty liver disease) with OMacor thErapy) http://www.clinicaltrials.gov registration number NCT00760513).]

It is beyond the scope of this review to discuss in detail advances in treatment for NAFLD. For a more detailed discussion of the efficacy of various treatments that have been tested in NAFLD see Bhatia et al. [3]. A summary of potential treatments for NAFLD is shown in Box 2.

Box 2

  1. Top of page
  2. Abstract
  3. Introduction
  4. Box 1
  5. Development of NAFLD
  6. Dietary lipid and carbohydrate and NAFLD
  7. How is NAFLD diagnosed?
  8. NAFLD and its association with Type 2 diabetes and cardiovascular disease
  9. Potential treatments for NAFLD
  10. Box 2
  11. Conclusions
  12. Competing interests
  13. Acknowledgements
  14. References
  15. Supporting Information

Potential treatments for non-alcoholic fatty liver disease (NAFLD)

Proven benefits

Weight loss where appropriate (decreased hepatic fat)

Improved fitness/increased physical activity (decreased hepatic fat)

Possible benefits

Pioglitazone (decreased hepatic fat in most but not all studies, decreased hepatic inflammation but no effect on hepatic inflammation)

Theoretical benefits

High dose n-3 polyunsaturated fatty acids

Rimonabant (cannabinoid receptor 1 blocker) (note: license now withdrawn)

Angiotensin II receptor blockers (theoretical benefit based on effect on fibrotic mechanisms in vitro)

Glucagon-like peptide 1 agonists (incretin effect to decrease appetite and facilitate weight loss, uncertain whether there is a direct effect on the liver)

Effect of adjunctive advice/treatments on the liver in NAFLD

‘Lifestyle’ treatments

Decreased alcohol intake should be recommended and potential abstinence from alcohol recommended [moderate alcohol intake (> 15 units per week in men) has a synergistic impact with obesity on the liver to increase risk of cirrhosis)

Drug effects on the liver

  •  Statins are safe and should be used where appropriate for cardiovascular risk reduction in this group of patients. Probably no benefit of this class on the liver in NAFLD
  •  Metformin is safe. Probably no benefit of this class on the liver in NAFLD
  •  Insulin promotes hepatic lipogenesis and may increase liver fat in the presence of increased carbohydrate intake

Conclusions

  1. Top of page
  2. Abstract
  3. Introduction
  4. Box 1
  5. Development of NAFLD
  6. Dietary lipid and carbohydrate and NAFLD
  7. How is NAFLD diagnosed?
  8. NAFLD and its association with Type 2 diabetes and cardiovascular disease
  9. Potential treatments for NAFLD
  10. Box 2
  11. Conclusions
  12. Competing interests
  13. Acknowledgements
  14. References
  15. Supporting Information

Non-alcoholic fatty liver disease (NAFLD) is an extremely common condition that is strongly associated with Type 2 diabetes and obesity. NAFLD is frequently a silent disease that is often diagnosed as a consequence of the further investigation of abnormal liver enzyme tests, noted as an incidental finding. NAFLD increases the risk of Type 2 diabetes and cardiovascular disease, and the combination of NAFLD, insulin resistance and central obesity occurs frequently in individuals. When all three risk factors (NAFLD, insulin resistance and obesity occur together), there is a marked increase in risk of Type 2 diabetes. NAFLD causes insulin resistance and this can make it difficult to achieve good glycaemic control in people who have diabetes. The pathogenesis of NAFLD is not fully elucidated and a better understanding of the factors causing liver fat, liver inflammation and liver fibrosis is needed. Increasing evidence suggests that altered fatty acid fluxes through the liver contribute to both liver fat and to liver inflammation, and these fluxes are potentially key to the initial development, and to progression, of NAFLD. It is likely that individual treatments will have varying effect on the different components of the NAFLD disease process. Consequently, new treatments need to be tested on liver fat, inflammation and fibrotic processes. Presently, there are no licensed treatments for NAFLD and therefore an emphasis needs to be placed on lifestyle advice, with weight loss where necessary, increases in physical activity and improved nutrition, in order to decrease liver fat accumulation.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Box 1
  5. Development of NAFLD
  6. Dietary lipid and carbohydrate and NAFLD
  7. How is NAFLD diagnosed?
  8. NAFLD and its association with Type 2 diabetes and cardiovascular disease
  9. Potential treatments for NAFLD
  10. Box 2
  11. Conclusions
  12. Competing interests
  13. Acknowledgements
  14. References
  15. Supporting Information

The author's research is supported in part by the NIHR (National Institute for Health Research) Southampton Biomedical Research Centre. Thanks to Lucinda England for helping with manuscript preparation and proofreading.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Box 1
  5. Development of NAFLD
  6. Dietary lipid and carbohydrate and NAFLD
  7. How is NAFLD diagnosed?
  8. NAFLD and its association with Type 2 diabetes and cardiovascular disease
  9. Potential treatments for NAFLD
  10. Box 2
  11. Conclusions
  12. Competing interests
  13. Acknowledgements
  14. References
  15. Supporting Information
  • 1
    Scorletti E, Calder PC, Byrne CD. Non-alcoholic fatty liver disease and cardiovascular risk: metabolic aspects and novel treatments. Endocrine 2011; 40: 332343.
  • 2
    Byrne CD, Olufadi R, Bruce KD, Cagampang FR, Ahmed MH. Metabolic disturbances in non-alcoholic fatty liver disease. Clin Sci (Lond) 2009; 116: 539564.
  • 3
    Bhatia LS, Curzen NP, Calder PC, Byrne CD. Non-alcoholic fatty liver disease: a new and important cardiovascular risk factor? Eur Heart J 2012; 33: 11901200.
  • 4
    Targher G, Day CP, Bonora E. Risk of cardiovascular disease in patients with nonalcoholic fatty liver disease. N Engl J Med 2010; 363: 13411350.
  • 5
    Sattar N, Scherbakova O, Ford I, O’Reilly DS, Stanley A, Forrest E et al. Elevated alanine aminotransferase predicts new-onset type 2 diabetes independently of classical risk factors, metabolic syndrome, and C-reactive protein in the West of Scotland Coronary Prevention Study. Diabetes 2004; 53: 28552860.
  • 6
    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: 11561161.
  • 7
    Ekstedt M, Franzen LE, Mathiesen UL, Thorelius L, Holmqvist M, Bodemar G et al. Long-term follow-up of patients with NAFLD and elevated liver enzymes. Hepatology 2006; 44: 865873.
  • 8
    Targher G, Bertolini L, Padovani R, Rodella S, Zoppini G, Pichiri I et al. Prevalence of non-alcoholic fatty liver disease and its association with cardiovascular disease in patients with type 1 diabetes. J Hepatol 2010; 53: 713718.
  • 9
    Holt HB, Wild SH, Wood PJ, Zhang J, Darekar AA, Dewbury K et al. Non-esterified fatty acid concentrations are independently associated with hepatic steatosis in obese subjects. Diabetologia 2006; 49: 141148.
  • 10
    Hodson L, Frayn KN. Hepatic fatty acid partitioning. Curr Opin Lipidol 2011; 22: 216224.
  • 11
    Byrne CD, Wareham NJ, Day NE, McLeish R, Williams DRR, Hales CN. Decreased non esterified fatty acid suppression and features of the insulin resistance syndrome occur in a sub-group of individuals with normal glucose tolerance. Diabetologia 1995; 38: 13581366.
  • 12
    Byrne CD, Wareham NJ, Brown DC, Clark PM, Cox LJ, Day NE et al. Hypertriglyceridaemia in subjects with normal and abnormal glucose tolerance: relative contributions of insulin secretion, insulin resistance and suppression of plasma non-esterified fatty acids. Diabetologia 1994; 37: 889896.
  • 13
    Bruce KD, Byrne CD. The metabolic syndrome: common origins of a multifactorial disorder. Postgrad Med J 2009; 85: 614621.
  • 14
    Bruce KD, Cagampang FR, Argenton M, Zhang J, Ethirajan PL, Burdge GC et al. Maternal high-fat feeding primes steatohepatitis in adult mice offspring, involving mitochondrial dysfunction and altered lipogenesis gene expression. Hepatology 2009; 50: 17961808.
  • 15
    Basciano H, Miller AE, Naples M, Baker C, Kohen R, Xu E et al. Metabolic effects of dietary cholesterol in an animal model of insulin resistance and hepatic steatosis. Am J Physiol Endocrinol Metab 2009; 297: E462E473.
  • 16
    Kleiner DE, Brunt EM, Van NM, Behling C, Contos MJ, Cummings OW et al. Design and validation of a histological scoring system for nonalcoholic fatty liver disease. Hepatology 2005; 41: 13131321.
  • 17
    Guha IN, Parkes J, Roderick PR, Harris S, Rosenberg WM. Non-invasive markers associated with liver fibrosis in non-alcoholic fatty liver disease. Gut 2006; 55: 16501660.
  • 18
    Rosenberg WM, Voelker M, Thiel R, Becka M, Burt A, Schuppan D et al. Serum markers detect the presence of liver fibrosis: a cohort study. Gastroenterology 2004; 127: 17041713.
  • 19
    Angulo P, Hui JM, Marchesini G, Bugianesi E, George J, Farrell GC et al. The NAFLD fibrosis score: a noninvasive system that identifies liver fibrosis in patients with NAFLD. Hepatology 2007; 45: 846854.
  • 20
    Ahmed MH BC. Non-alcoholic steatatohepatitis and metabolic syndrome. In: Byrne C, Wild S, eds. Metabolic Syndrome. Chichester: John Wiley and Sons, 2005: 279305.
  • 21
    Ahmed MH BC. Metabolic syndrome, diabetes and CHD risk. In: Packard CJ, ed. The Year in Lipid Disorders. Oxford: Clinical Publishing, 2007: 326.
  • 22
    Bedogni G, Miglioli L, Masutti F, Tiribelli C, Marchesini G, Bellentani S. Prevalence of and risk factors for nonalcoholic fatty liver disease: the Dionysos Nutrition and Liver Study. Hepatology 2005; 42: 4452.
  • 23
    Bellentani S, Saccoccio G, Masutti F, Croce LS, Brandi G, Sasso F et al. Prevalence of and risk factors for hepatic steatosis in Northern Italy. Ann Intern Med 2000; 132: 112117.
  • 24
    Rocha R, Cotrim HP, Carvalho FM, Siqueira AC, Braga H, Freitas LA. Body mass index and waist circumference in non-alcoholic fatty liver disease. J Hum Nutr Diet 2005; 18: 365370.
  • 25
    Pagano G, Pacini G, Musso G, Gambino R, Mecca F, Depetris N et al. Nonalcoholic steatohepatitis, insulin resistance, and metabolic syndrome: further evidence for an etiologic association. Hepatology 2002; 35: 367372.
  • 26
    Chitturi S, Abeygunasekera S, Farrell GC, Holmes-Walker J, Hui JM, Fung C et al. NASH and insulin resistance: insulin hypersecretion and specific association with the insulin resistance syndrome. Hepatology 2002; 35: 373379.
  • 27
    Sung KC, Jeong WS, Wild SH, Byrne CD. Combined influence of insulin resistance, overweight/obesity, and fatty liver as risk factors for Type 2 diabetes. Diabetes Care 2012; 35: 717722.
  • 28
    Gastaldelli A, Kozakova M, Hojlund K, Flyvbjerg A, Favuzzi A, Mitrakou A et al. Fatty liver is associated with insulin resistance, risk of coronary heart disease, and early atherosclerosis in a large European population. Hepatology 2009; 49: 15371544.
  • 29
    Yun KE, Shin CY, Yoon YS, Park HS. Elevated alanine aminotransferase levels predict mortality from cardiovascular disease and diabetes in Koreans. Atherosclerosis 2009; 205: 533537.
  • 30
    Targher G, Bertolini L, Rodella S, Tessari R, Zenari L, Lippi G et al. Nonalcoholic fatty liver disease is independently associated with an increased incidence of cardiovascular events in type 2 diabetic patients. Diabetes Care 2007; 30: 21192121.
  • 31
    Soderberg C, Stal P, Askling J, Glaumann H, Lindberg G, Marmur J et al. Decreased survival of subjects with elevated liver function tests during a 28-year follow-up. Hepatology 2010; 51: 595602.
  • 32
    Schindhelm RK, Dekker JM, Nijpels G, Bouter LM, Stehouwer CD, Heine RJ et al. Alanine aminotransferase predicts coronary heart disease events: a 10-year follow-up of the Hoorn Study. Atherosclerosis 2007; 191: 391396.
  • 33
    Ruttmann E, Brant LJ, Concin H, Diem G, Rapp K, Ulmer H. Gamma-glutamyltransferase as a risk factor for cardiovascular disease mortality: an epidemiological investigation in a cohort of 163 944 Austrian adults. Circulation 2005; 112: 21302137.
  • 34
    Lee DH, Silventoinen K, Hu G, Jacobs DR Jr, Jousilahti P, Sundvall J et al. Serum gamma-glutamyltransferase predicts non-fatal myocardial infarction and fatal coronary heart disease among 28 838 middle-aged men and women. Eur Heart J 2006; 27: 21702176.
  • 35
    Haring R, Wallaschofski H, Nauck M, Dorr M, Baumeister SE, Volzke H. Ultrasonographic hepatic steatosis increases prediction of mortality risk from elevated serum gamma-glutamyl transpeptidase levels. Hepatology 2009; 50: 14031411.
  • 36
    Hamaguchi M, Kojima T, Takeda N, Nagata C, Takeda J, Sarui H et al. Nonalcoholic fatty liver disease is a novel predictor of cardiovascular disease. World J Gastroenterol 2007; 13: 15791584.
  • 37
    Fraser A, Harris R, Sattar N, Ebrahim S, Smith GD, Lawlor DA. Gamma-glutamyltransferase is associated with incident vascular events independently of alcohol intake: analysis of the British Women’s Heart and Health Study and Meta-Analysis. Arterioscler Thromb Vasc Biol 2007; 27: 27292735.
  • 38
    Dunn W, Xu R, Wingard DL, Rogers C, Angulo P, Younossi ZM et al. Suspected nonalcoholic fatty liver disease and mortality risk in a population-based cohort study. Am J Gastroenterol 2008; 103: 22632271.
    Direct Link:
  • 39
    Adams LA, Lymp JF, St Sauver J, Sanderson SO, Lindor KD, Feldstein A et al. The natural history of nonalcoholic fatty liver disease: a population-based cohort study. Gastroenterology 2005; 129: 113121.
  • 40
    Ahmed MH, Byrne CD. Current treatment of non-alcoholic fatty liver disease. Diabetes Obes Metab 2009; 11: 188195.
  • 41
    Perseghin G. The role of non-alcoholic fatty liver disease in cardiovascular disease. Dig Dis 2010; 28: 210213.
  • 42
    Bhala N, Angulo P, van der Poorten D, Lee E, Hui JM, Saracco G et al. The natural history of nonalcoholic fatty liver disease with advanced fibrosis or cirrhosis: an international collaborative study. Hepatology 2011; 54: 12081216.
  • 43
    Sung KC, Ryan MC, Wilson AM. The severity of nonalcoholic fatty liver disease is associated with increased cardiovascular risk in a large cohort of non-obese Asian subjects. Atherosclerosis 2009; 203: 581586.
  • 44
    McKimmie RL, Daniel KR, Carr JJ, Bowden DW, Freedman BI, Register TC et al. Hepatic steatosis and subclinical cardiovascular disease in a cohort enriched for type 2 diabetes: the Diabetes Heart Study. Am J Gastroenterol 2008; 103: 30293035.
    Direct Link:
  • 45
    Dam-Larsen S, Becker U, Franzmann MB, Larsen K, Christoffersen P, Bendtsen F. Final results of a long-term, clinical follow-up in fatty liver patients. Scand J Gastroenterol 2009; 44: 12361243.
  • 46
    Alkhouri N, Tamimi TA, Yerian L, Lopez R, Zein NN, Feldstein AE. The inflamed liver and atherosclerosis: a link between histologic severity of nonalcoholic fatty liver disease and increased cardiovascular risk. Dig Dis Sci 2010; 55: 26442650.
  • 47
    Ramilli S, Pretolani S, Muscari A, Pacelli B, Arienti V. Carotid lesions in outpatients with nonalcoholic fatty liver disease. World J Gastroenterol 2009; 15: 47704774.
  • 48
    McCullough AJ. The clinical features, diagnosis and natural history of nonalcoholic fatty liver disease. Clin Liver Dis 2004; 8: 521533, viii.
  • 49
    Tolman KG, Fonseca V, Tan MH, Dalpiaz A. Narrative review: hepatobiliary disease in type 2 diabetes mellitus. Ann Intern Med 2004; 141: 946956.
  • 50
    Jousilahti P, Rastenyte D, Tuomilehto J. Serum gamma-glutamyl transferase, self-reported alcohol drinking, and the risk of stroke. Stroke 2000; 31: 18511855.
  • 51
    Wannamethee G, Ebrahim S, Shaper AG. Gamma-glutamyltransferase: determinants and association with mortality from ischemic heart disease and all causes. Am J Epidemiol 1995; 142: 699708.
  • 52
    Schindhelm RK, Dekker JM, Nijpels G, Bouter LM, Stehouwer CD, Heine RJ et al. Alanine aminotransferase predicts coronary heart disease events: a 10-year follow-up of the Hoorn Study. Atherosclerosis 2007; 191: 391396.
  • 53
    Monami M, Bardini G, Lamanna C, Pala L, Cresci B, Francesconi P et al. Liver enzymes and risk of diabetes and cardiovascular disease: results of the Firenze Bagno a Ripoli (FIBAR) study. Metabolism 2008; 57: 387392.
  • 54
    Targher G, Bertolini L, Padovani R, Rodella S, Tessari R, Zenari L et al. Prevalence of nonalcoholic fatty liver disease and its association with cardiovascular disease among type 2 diabetic patients. Diabetes Care 2007; 30: 12121218.
  • 55
    Targher G, Bertolini L, Poli F, Rodella S, Scala L, Tessari R et al. Nonalcoholic fatty liver disease and risk of future cardiovascular events among type 2 diabetic patients. Diabetes 2005; 54: 35413546.
  • 56
    Ahmed MH, Byrne CD. Ezetimibe as a potential treatment for non-alcoholic fatty liver disease: is the intestine a modulator of hepatic insulin sensitivity and hepatic fat accumulation? Drug Discov Today 2010; 15: 590595.
  • 57
    Calder PC. The relationship between the fatty acid composition of immune cells and their function. Prostaglandins Leukot Essent Fatty Acids 2008; 79: 101108.
  • 58
    Riediger ND, Othman RA, Suh M, Moghadasian MH. A systemic review of the roles of n-3 fatty acids in health and disease. J Am Diet Assoc 2009; 109: 668679.
  • 59
    Calder PC, Yaqoob P. Understanding omega-3 polyunsaturated fatty acids. Postgrad Med 2009; 121: 148157.
  • 60
    Kotronen A, Yki-Jarvinen H. Fatty liver. A novel component of the metabolic syndrome. Arterioscler Thromb Vasc Biol 2008; 28: 2738.
  • 61
    Araya J, Rodrigo R, Videla LA, Thielemann L, Orellana M, Pettinelli P et al. Increase in long-chain polyunsaturated fatty acid n-6/n-3 ratio in relation to hepatic steatosis in patients with non-alcoholic fatty liver disease. Clin Sci (Lond) 2004; 106: 635643.
  • 62
    Reddy JK. Nonalcoholic steatosis and steatohepatitis. III. Peroxisomal β-oxidation, PPAR-α, and steatohepatitis. Am J Physiol Gastrointest Liver Physiol 2001; 281: G1333G1339.
  • 63
    Clarke SD. Polyunsaturated fatty acid regulation of gene transcription: a molecular mechanism to improve the metabolic syndrome. J Nutr 2001; 131: 11291132.
  • 64
    Clarke SD. Nonalcoholic steatosis and steatohepatitis. I. Molecular mechanism for polyunsaturated fatty acid regulation of gene transcription. Am J Physiol Gastrointest Liver Physiol 2001; 281: G865G869.
  • 65
    Clarke SD. The multi-dimensional regulation of gene expression by fatty acids: polyunsaturated fats as nutrient sensors. Curr Opin Lipidol 2004; 15: 1318.

Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Box 1
  5. Development of NAFLD
  6. Dietary lipid and carbohydrate and NAFLD
  7. How is NAFLD diagnosed?
  8. NAFLD and its association with Type 2 diabetes and cardiovascular disease
  9. Potential treatments for NAFLD
  10. Box 2
  11. Conclusions
  12. Competing interests
  13. Acknowledgements
  14. References
  15. Supporting Information

Data S1.Power Point Presentation

FilenameFormatSizeDescription
DME_3732_sm_AppendixS1.ppt5235KSupporting info item

Please note: Wiley Blackwell is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.