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Clinical Science (2009) 116, 539–564 (Printed in Great Britain) doi:10.1042/CS20080253 539


Metabolic disturbances in non-alcoholic

fatty liver disease

Christopher D. BYRNE∗ , Rasaq OLUFADI†, Kimberley D. BRUCE∗ ,

Felino R. CAGAMPANG‡ and Mohamed H. AHMED†

Endocrinology & Metabolism Unit, Institute for Developmental Sciences, University of Southampton and Southampton
University Hospitals Trust, Southampton General Hospital, Southampton SO16 6YD, U.K., †Chemical Pathology Department,
Southampton University Hospitals Trust, Southampton General Hospital, Southampton SO16 6YD, U.K., and ‡Centre for
Developmental Origins of Health and Disease, Department of Maternal, Fetal & Neonatal Physiology, University of

Clinical Science
Southampton School of Medicine, Princess Anne Hospital, Southampton SO16 5AY, U.K.


NAFLD (non-alcoholic fatty liver disease) refers to a wide spectrum of liver damage, ranging from
simple steatosis to NASH (non-alcoholic steatohepatitis), advanced fibrosis and cirrhosis. NAFLD
is strongly associated with insulin resistance and is defined by accumulation of liver fat > 5 %
per liver weight in the presence of < 10 g of daily alcohol consumption. The exact prevalence of
NAFLD is uncertain because of the absence of simple non-invasive diagnostic tests to facilitate
an estimate of prevalence. In certain subgroups of patients, such as those with Type 2 diabetes,
the prevalence of NAFLD, defined by ultrasound, may be as high as 70 %. NASH is an important
subgroup within the spectrum of NAFLD that progresses over time with worsening fibrosis and
cirrhosis, and is associated with increased risk for cardiovascular disease. It is, therefore, important
to understand the pathogenesis of NASH and, in particular, to develop strategies for inter-
ventions to treat this condition. Currently, the ‘gold standard’ for the diagnosis of NASH is liver
biopsy, and the need to undertake a biopsy has impeded research in subjects in this field. Limited
results suggest that the prevalence of NASH could be as high as 11 % in the general population,
suggesting there is a worsening future public health problem in this field of medicine. With a
burgeoning epidemic of diabetes in an aging population, it is likely that the prevalence of NASH
will continue to increase over time as both factors are important risk factors for liver fibrosis.
The purpose of this review is to: (i) briefly discuss the epidemiology of NAFLD to describe the
magnitude of the future potential public health problem; and (ii) to discuss extra- and intra-hepatic
mechanisms contributing to the pathogenesis of NAFLD, a better understanding of which may
help in the development of novel treatments for this condition.

Key words: diabetes, inflammation, mitochondrion, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis
(NASH), obesity.
Abbreviations: ACC, acetyl-CoA carboxylase; ALT, alanine aminotransferase; AMPK, AMP-activated kinase; AP-1, activated
protein-1; aRR, adjusted relative risk; ASO, antisense oligonucleotide; AST, aspartate aminotransferase; BMI, body mass index;
BP, blood pressure; CCT, CTP:phosphocholine cytidylyltransferase; CHD, coronary heart disease; ChREBP, carbohydrate-
responsive-element-binding protein; CI, confidence interval; CKD, chronic kidney disease; CPT, CDP-choline:1,2 diacylglycerol
choline phosphotransferase; CPT-1, carnitine palmitoyltransferase-1; CVD, cardiovascular disease; DAG, diacylglycerol; DGAT-2,
diacylglycerol acyltransferase-2; ELF score, European Liver Fibrosis score; ER, endoplasmic reticulum; FAS, fatty acid synthase;
Fox, forkhead box; FRS, Framingham risk score; GFR, glomerular filtration rate; GGT, γ -glutamyltransferase; GLUT4, glucose
transporter 4; HbA1c , glycated haemoglobin; HDL, high-density lipoprotein; HSL, hormone-sensitive lipase; IL, interleukin; IMT,
intima-media thickness; INSIG-1, insulin induced gene-1; IRE-1, inositol-requiring enzyme-1; IRS, insulin receptor substrate; IU,
international units; JNK, c-Jun N-terminal kinase; LDL, low-density lipoprotein; L-PK, liver-specific pyruvate kinase; LPS, lipo-
polysaccharide; LXR-α, liver X receptor-α; MAPK, mitogen-activated protein kinase; MCD, methionine- and choline-deficient;
MRI, magnetic resonance imaging; MRS, magnetic resonance spectroscopy; mtDNA, mitochondrial DNA; mTOR, mammalian
target of rapamycin; MUFA, mono-unsaturated fatty acid; NAFLD, non-alcoholic fatty liver disease; NASH, non-alcoholic
steatohepatitis; NEFA, non-esterified fatty acid (‘free fatty acid’); NF-κB, nuclear factor κB; (continued overleaf)
Correspondence: Professor Christopher D. Byrne (email

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540 C. D. Byrne and others

NAFLD (non-alcoholic fatty liver disease) refers to a NAFLD is very common and occurs in individuals of all
wide spectrum of liver damage, ranging from simple ste- ages and ethnic groups. Adjusted prevalence of NAFLD
atosis to steatohepatitis, advanced fibrosis and cirrhosis. using MRS (magnetic resonance spectroscopy) in 2287
NAFLD is strongly associated with insulin resistance individuals (U.S.A.) was found to be approx. 34 %, and
and is defined by accumulation of liver fat > 5 % per 90 % of these cases of NAFLD were attributed to non-
liver weight in the presence of < 10 g of daily alcohol alcoholic causes [2]. Furthermore, the Dallas Heart Study,
consumption. The characteristic histology of NAFLD which was a population-based cohort study performed
resembles that of alcohol-induced liver injury, but occurs in an ethnically diverse community in U.S.A. using
in people who consume minimal alcohol. NAFLD is MRS, reported that the prevalence of NAFLD is approx.
regarded as the most common cause of increased liver 33.6 % [3]. On the other hand, the use of ultrasound in
enzymes in the U.S.A., associated with Type 2 diabetes, the Dionysos nutrition and liver disease study in Italy
obesity and hyperlipidaemia [1]. The reported prevalence found that the prevalence of NAFLD was approx. 25 %
of obesity with NAFLD varies between 30 and 100 %, and was associated with most features of the metabolic
whereas the prevalence of NAFLD within Type 2 diabetes syndrome [4]. Findings from the same study showed that
varies between 10 and 75 %. In routine clinical practice, the prevalence of steatosis was increased in heavy drinkers
most cases of fatty liver disease are attributable to {46.4 % [95 % CI (confidence interval), 34–59%]} and
alcohol excess; however, fatty liver disease can also obese persons [75.8 % (95 % CI, 63–85 %)] compared
occur in association with a wide range of toxins, drugs with controls [16.4 % (95 % CI, 8–25 %)]. Steatosis was
and diseases, such as morbid obesity, cachexia, Type 2 found in 94.5 % (95 % CI, 85–99 %) of obese heavy drink-
diabetes, hyperlipidaemia and after jejunoileal bypass ers. Compared with controls, the risk for steatosis was
surgery. As important risk factors for NAFLD, such as 2.8-fold higher (95 % CI, 1.4–7.1-fold) in heavy drinkers,
obesity and Type 2 diabetes, are increasing in prevalence 4.6-fold (95 % CI, 2.5–11.0-fold) in obese people, and 5.8-
it is likely that there will be a marked increase in NAFLD fold (95 % CI, 3.2–12.3-fold) in people who were obese
with important consequences for healthcare providers. and drank heavily. In heavy drinkers, obesity increased
NAFLD is rapidly becoming an important public the risk of steatosis 2-fold (95 % CI, 1.5–3.0-fold;
health problem. Undiagnosed, this condition may pro- P < 0.001), but heavy drinking was associated with only a
gress silently and results in cirrhosis, portal hypertension 1.3-fold (95 % CI, 1.02–1.6-fold) increase in risk in obese
and liver-related death in early adulthood. Interestingly, persons (P = 0.0053). It was concluded that steatosis is
NAFLD is a hepatic component of the metabolic frequently encountered in healthy persons and is usually
syndrome and is an independent risk factor for CVD present in obese persons who drink more than 60 g of
(cardiovascular disease). Importantly, NAFLD is associ- alcohol per day. Steatosis is more strongly associated
ated with an increased risk of all-cause death and predicts with obesity than with heavy drinking, suggesting a
future CVD events, independently of age, gender, LDL greater role of overweight than alcohol consumption in
(low-density lipoprotein)-cholesterol, smoking and the accumulation of fat in the liver [4]. It is likely that there is
cluster of features of the metabolic syndrome. Currently, an additive effect of obesity and alcohol consumption to
there are no biochemical markers for NAFLD, and an worsen the NAFLD phenotype. Moreover, these findings
increase (or decrease) in ALT (alanine aminotransferase) raise important questions about the safety of any alcohol
is often used as a biochemical marker to monitor consumption in obese people who are insulin-resistant
progression (or amelioration) of NAFLD (despite the because obesity and alcohol are both likely to contribute
fact that ALT concentrations can be misleading and do to the NAFLD phenotype. Importantly, the prevalence
not reflect the severity or the outcome). Mass screening of NAFLD as determined by ultrasound increased from
for significant liver injury in patients with NAFLD will 16.4 % among individuals with normal BMI (body mass
be an important medical challenge in the years to come index) to 75.8 % among obese people. The prevalence
because of the epidemics of obesity and diabetes. of NAFLD is even higher with morbid obesity and

Abbreviations cont.
IκB, inhibitor of NF-κB; IKK, IκB kinase; NEMO, NF-κB essential modulator; OR, odds ratio; PC, phosphatidylcholine;
PE, phosphatidylethanolamine; PEMT, phosphatidylethanolamine N-methyltransferase; PERK, PKR (double-stranded-RNA-
dependent protein kinase)-like ER kinase; PI3K, phosphoinositide 3-kinase; PKC, protein kinase C; PP2A, protein phosphatase
2A; PPAR, peroxisome-proliferator-activated receptor; PTEN, phosphatase and tensin homologue deleted on chromosome 10;
PUFA, polyunsaturated fatty acid; ROS, reactive oxygen species; SAM, S-adenosylmethionine; SCD-1, stearoyl-CoA desaturase-1;
SHIP-2, SH2-containing inositol phosphatase-2; SREBP-1c, sterol-regulatory-element binding-protein-1c; TAG, triacylglycerol;
TGF, transforming growth factor; TNF-α, tumour necrosis factor-α; UDCA, ursodeoxycholic acid; VLDL, very-LDL.

C The Authors Journal compilation 
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Metabolic disturbances in non-alcoholic fatty liver disease 541

among morbidly obese undergoing bariatric surgery the the early observation of both the Danish and Dionyosis
prevalence may be as high as 96 % [5]. Interestingly, studies that simple steatosis had benign natural history.
the prevalence of NAFLD in the Japanese population Generally speaking, the natural history of NASH (non-
rises with increasing degrees of hyperglycaemia, being alcoholic steatohepatitis) is associated with the possibility
approx. 27 % in people with normal fasting glucose of progression to cirrhosis. Progression of liver fibrosis
levels, increasing to 43 % among those with impaired was found in one-third of 106 NASH patients 4.3 years
fasting glycaemia, and 62 % among newly diagnosed after the first liver biopsy, and obesity and BMI were
diabetes [6]. On the other hand, in non-diabetic and the only associated factors with such progression [19].
non-obese individuals, the OR (odds ratio) for metabolic Furthermore, in a large study of the natural history of
disorders (insulin resistance, hypertriacylglycerolaemia the disease, follow-up biopsies were performed after
and hyperuricaemia) in subjects with NAFLD, compared 12–20 years in 772 patients with NAFLD [20]. Of
with those without NAFLD in the normal-weight group, 132 patients for whom data were complete, 4 % of sub-
was higher than that in the overweight group. Multiple jects with fatty liver progressed to cirrhosis compared
logistic regression analysis showed that gender, waist with 22 % of patients with NASH and fibrosis on the first
circumference, TAG [triacylglycerol (triglyceride)] level biopsy [20]. All liver-related morbidity and mortality
and insulin resistance were independently associated with were also noted in the later group with NASH, rather than
NAFLD in the normal-weight group [7]. simple hepatic steatosis. Thus the evidence, albeit rather
Among the Asian population (known to have high limited, suggest that NASH is not a harmless condition.
visceral fat), the prevalence of NAFLD using ultrasound Importantly, Adams et al. [18] showed that the 8 % liver-
has been estimated to vary between 5 and 40 % [8]. related mortality in biopsy-proven NASH is similar to
In Japan, it has been estimated that the prevalence of the 10 % mortality reported by Matteoni and co-workers
increased ALT attributable to NAFLD has increased [20], and there is general agreement that once cirrhosis
from less than 10 % in 1984 to 25 % in 2001 [9]. In China, develops in patients with NAFLD the prognosis is poor
the prevalence of NAFLD has been estimated to vary [18–23].
from 5 to 24 %, and this variation may be attributed to
lifestyle differences between rural and urban populations
[10,11]. An increase in the prevalence of obesity and DIAGNOSIS AND BIOCHEMICAL MARKERS
diabetes may also have resulted in an increase in the
prevalence of NAFLD in the Middle East. However, in At present, a liver biopsy remains the only reliable
a study by el-Hassan et al. [12] in 1992, the prevalence of way to diagnose NAFLD and establish the presence of
NAFLD was approx. 10 %, whereas in contrast a small fibrosis. The sampling variability has the potential to
study in diabetic Saudi individuals has suggested the alter significantly the diagnosis and staging of NAFLD.
prevalence of NAFLD is approx. 55 % [13]. It is not practical to offer liver biopsy as a test for the
Children may also develop NAFLD. In a retrospective diagnosis of NAFLD, as the prevalence of the condition
review of 742 children between the ages of 2 and 19 years would overwhelm service provision. Consequently
who had an autopsy performed from 1993 to 2003, fatty non-invasive markers of NAFLD are urgently needed
liver was present in 13 % of subjects [14]. In a small that can be used for diagnosis and monitoring responses
study of 44 obese children, aged 6–16 years, with a BMI to therapy. Table 1 shows a summary of biochemical
above the 97th centile, hepatic fat content was measured markers and the potential advantages and disadvantages
by phase-contrast MRI (magnetic resonance imaging) of different tests for identification of NAFLD. As can
and an increased hepatic fat fraction was identified in been seen from Table 1, no single biochemical test has,
14 subjects (31.8 %) [15]. to date, shown sufficient sensitivity and specificity to be
There are few studies of the natural history of NAFLD; used in clinical practice for the diagnosis or monitoring of
however, recent findings strongly suggest NAFLD is not NAFLD.
a harmless condition and depends critically on disease It is beyond the scope of this review to discuss
stage. A Danish study (215 patients with biopsy-proven biomarkers for NAFLD in detail; however, it is
NAFLD) concluded that NAFLD has a benign course important to realize that many studies investigating the
without excess mortality [16]. This view was endorsed aetiology and pathogenesis and investigating potential
further by 10-year follow-up data from the Dionyosis treatments for NAFLD have inevitably not been able
study [17]. However, a recent large study by Adams et al. to use liver biopsy, but have used proxy markers for
[18] in 420 patients with NAFLD, between 1980 and the disease. This unfortunate problem has made it very
2000, concluded that the overall death rate is higher than difficult to establish which risk factors are aetiologically
expected [standardized mortality ratio, 1.34 (95 % CI, linked to the different components of the liver disease and
1.003–1.76); P = 0.03], and this increase in mortality was which treatments improve liver fat, or liver inflammation
associated with age, impaired fasting glucose/diabetes or liver fibrosis within the spectrum of NAFLD.
and cirrhosis. Importantly, Adams et al. [18] confirmed To date, these difficulties have impeded progress in

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542 C. D. Byrne and others

Table 1 Summary of the use of simple biochemical markers in NAFLD

CDT, carbohydrate-deficient transferrin; CK-18, cytokeratin 18; hsCRP, high-sensitivity C-reactive protein; oxLDL, oxidized LDL; RT–PCR, reverse transcriptase–PCR; TBARS,
thiobarbituric acid-reacting substances.

Biochemical marker Potential advantages and disadvantages References

ALT Treatment with UDCA and vitamin E decreased serum ALT, but failed to show a parallel improvement in [215–217]
histology. Importantly, the entire histological pattern of NAFLD can be seen in the presence of normal
ALT. The sensitivity of ALT for the diagnosis of NASH in large cohort was found to be only 40 %.
AST/ALT ratio Relatively poor sensitivity and specificity in NAFLD. [218]
GGT Increased with NAFLD; the increase is also seen with high alcohol intake. [1]
hsCRP Increased in patients with histologically verified NAFLD. Multiple regression analysis revealed that in [219,220]
comparison with cases of steatosis, hsCRP was significantly increased in cases of NASH and fibrosis. The
results of the RT–PCR showed that intrahepatic mRNA expression of CRP was increased in NAFLD and
TNF-α and adiponectin TNF-α is increased in NAFLD, whereas adiponectin is decreased. A low adiponectin level in NAFLD patients [1]
was reported and may represent a pathogenic mechanism in NAFLD. High adiponectin levels have been
reported to protect against both alcoholic fatty liver disease and NAFLD. Further studies are needed
before adiponectin can be utilized as a biochemical marker for NAFLD.
NEFAs Increased NEFA concentrations are associated with NAFLD independently of classic measures of insulin [221]
resistance. Elevated NEFA concentrations have been associated with impaired glucose utilization,
impaired suppression of glucose production and impaired insulin secretion; however, there are problems
in utilizing NEFA concentrations as a biochemical marker for NAFLD because of the marked variation in
levels throughout the day.
Impaired glucose tolerance In two large separate studies of patients undergoing anti-obesity surgery (551 patients by Marceau et al. [18,63,222,223]
[63] and 505 patients by Luyckx et al. [223]), the severity of NAFLD was closely related to impaired
glycaemia. Haukeland et al. [222] concluded that abnormal glucose tolerance independently predicts
both fatty liver and fibrosis in 88 patients. Adams et al. [18], in a study of 420 patients with NAFLD,
showed that the overall death rate in patients with NAFLD is higher than expected, especially those with
impaired fasting glucose or diabetes.
oxLDL and TBARS Unreliable biochemical markers for NAFLD. [224]
CK-18 CK-18 is a major intermediate filament protein in the liver which is associated with morphological changes [225]
in apoptosis and is increased in NAFLD. Sensitivity and specificity of CK-18 in NAFLD has been reported
to be more than 90 %, and CK-18 was independently associated with NASH in multivariate analysis.
CDT Used to differentiate between NASH and alcoholic hepatitis. [226]
Apolipoprotein A1, haptoglobin, No markers are specific for NAFLD. [227]
a2 -macroglobulin, hyaluronic
acid and procollagen peptides,
such as I, III and VI

understanding the aetiology of NAFLD in humans and showing considerable promise with good sensitivity and
have hampered progress in assessing new treatments for specificity for NASH. A concentrated research initiative
the condition. Despite these problems, progress is being is now needed to address the pressing need of sensitive
made in developing non-invasive diagnostic markers for specific tests for NAFLD. The challenge remains to
investigating aetiology and for testing new treatments establish biomarkers that are simple, reproducible,
for NAFLD. Recently, the ELF score (European Liver inexpensive and with high sensitivity and specificity
Fibrosis score) has been tested in a small subgroup of for NAFLD and can differentiate simple steatosis from
patients with NAFLD recruited with liver fibrosis [24]. NASH. Identification of simple inexpensive biomarkers
The ELF score comprises three simple biochemical would facilitate achieving reliable estimates of prevalence
measurements fitted into a proprietary algorithm and worldwide and would provide diagnostic tools for the
shows promise with good sensitivity and specificity for monitoring of responses to therapeutic interventions.
NASH with fibrosis, although further research is needed. Ultrasound, CT (computer tomography) scanning
Another simpler algorithm has recently been developed and MRI have all been used in the diagnosis of NAFLD.
and published in full utilizing simple anthropometric Ultrasound has a sensitivity of 89 % and specificity
measurements and biochemical tests [25]. This test is also of 77 % and is commonly used in a clinical practice

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Metabolic disturbances in non-alcoholic fatty liver disease 543

setting. Quantitative assessment of fatty infiltration is of the metabolic syndrome [33], suggesting that NAFLD
best achieved with MRI [1]. is associated with CHD independently of other features
of the metabolic syndrome.
Interestingly, another study from the U.S.A. has
RELATIONSHIP WITH CVD examined the association between elevated serum ALT
activity and the 10-year risk of CHD as estimated
Recent findings now suggests that NAFLD may also using the FRS (Framingham risk score) [34]. Among
be linked to increased CVD risk in Type 2 diabetes. participants without viral hepatitis or excessive alcohol
CVD risk varies markedly between individuals with consumption, those with increased ALT activity [> 43 IU
Type 2 diabetes and we have shown that, using traditional (international units)/l] (n = 267) had a higher FRS than
algorithms for risk prediction, CVD events may be under- those with normal ALT activity (n = 7259), both among
estimated by approximately one-third in Type 2 diabetes men [mean difference in FRS, 0.25 (95 % CI, 0.07–0.4);
[26]. We have also shown that there is a > 4-fold and hazard ratio for CHD, 1.28 (95 % CI, 1.07–1.5)] and
statistically significant increase in risk of CHD (coronary women [mean difference in FRS, 0.76 (95 % CI, 0.4–1.1);
heart disease) death if patients have all five features of hazard ratio for CHD, 2.14 (95 % CI, 1.5–3.0)]. The
the metabolic syndrome [central obesity, high TAG, ALT threshold for an increased risk of CHD was
low HDL (high-density lipoprotein)-cholesterol, high higher in men (> 43 IU/l) than in women (> 30 IU/l)
BP (blood pressure) and insulin resistance] compared [34]. Recently, the FIBAR (Firenze Bagno a Ripoli)
with having only one feature (e.g. hyperglycaemia in study concluded that increased GGT or AST (aspartate
isolation) [27]. Importantly, given the heterogeneity of aminotransferase) is an independent predictor of CVD.
cardiovascular risk in people with Type 2 diabetes, we An increase in GGT levels above the reference range, or
have shown that patients with NAFLD and obesity are also in the upper reference range, was also an independent
markedly more insulin-resistant in muscle and fat than predictor of incident diabetes [35].
those subjects with obesity alone [28]. In a recent study by Targher et al. [36] (n = 2839)
Prospective studies have reported associations between NAFLD and CVD were the main outcome measures in
increased liver enzymes [particularly the serum GGT Type 2 diabetic patients. The authors showed that the
(γ -glutamyltransferase) level as surrogate markers of unadjusted prevalence of NAFLD was 69.5 % among
NAFLD) [29,30] and the occurrence of CVD events in participants, and NAFLD was the most common cause
both non-diabetic subjects and Type 2 diabetic patients. (81.5 %) of hepatic steatosis detected by ultrasound.
In a study of 14 874 middle-aged Finnish men and women, The prevalence of NAFLD increased with age (65.4 %
a small increase in GGT levels were independently among participants aged 40–59 years, and 74.6 % among
associated with an increased risk of ischaemic stroke in those aged  60 years; P < 0.001) and the age-adjusted
both sexes [31]. Among 7613 middle-aged British men prevalence of NAFLD was 71.1 % in men and 68 % in
followed for 11.5 years, increased GGT levels were inde- women. NAFLD patients had a higher age (P < 0.001)
pendently associated with a significant increase in mortal- and gender-adjusted prevalence of coronary (26.6 com-
ity from all causes and from CHD [32]. Recent epidemio- pared with 18.3 %), cerebrovascular (20.0 compared
logical studies have suggested the increase in ALT may be with 13.3 %) and peripheral (15.4 compared with
linked to increase in risk of CVD. The Hoorn Study is a 10.0 %) vascular disease than their counterparts without
population-based cohort of Caucasian men and women NAFLD. In logistic regression analysis, NAFLD was
aged 50–75 years at baseline. The 10-year risk of all- associated with prevalent CVD, independent of classical
cause mortality, fatal and non-fatal CVD and CHD risk factors, glycaemic control, medication and the
events in relation to ALT baseline was assessed in 1439 features of the metabolic syndrome [36].
subjects [33]. Subjects with prevalent CVD/CHD and The Valpolicella Heart Diabetes Study is a prospective
missing data were excluded from these analyses. When nested case-control study in 2103 Type 2 diabetic
compared with the first tertile, the age- and gender- patients who were free of diagnosed CVD at baseline.
adjusted hazard ratios (95 % CIs) for all-cause mortality, During 5 years of follow-up, 248 participants (case
CVD events and CHD events were 1.30 (0.92–1.83), subjects) subsequently developed non-fatal CHD
1.40 (1.09–1.81) and 2.04 (1.35–3.10) respectively, for (myocardial infarction and coronary revascularization
subjects in the upper tertile of ALT. After adjustment for procedures), ischaemic stroke or cardiovascular death.
components of the metabolic syndrome and traditional After adjustment for age, gender, smoking history,
risk factors, the association between ALT and CHD diabetes duration, HbA1c (glycated haemoglobin),
events remained significant for subjects in the third LDL-cholesterol, liver enzymes and use of medication,
tertile relative to those in the first tertile, with a hazard the presence of NAFLD was significantly associated
ratio (95 % CI) of 1.88 (1.21–2.92). Thus the Hoorn with an increase in CVD risk [OR, 1.84 (95 % CI, 1.4–
Study shows that ALT predicts cardiovascular events 2.1); P < 0.001]. Additional adjustment for the metabolic
independently of traditional risk factors and the features syndrome (as defined by National Cholesterol Education

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544 C. D. Byrne and others

Program Adult Treatment Panel III criteria) appreciably Interestingly, NAFLD has been shown to be associated
attenuated, but did not abolish, this association [OR, with the development of CKD in Korean individuals
1.53 (95 % CI, 1.1–1.7); P = 0.02] [37]. Thus the above [crude relative risk, 2.18 (95 % CI, 1.75–2.71], and this
two large studies have demonstrated that NAFLD is relationship remained significant even after adjustment
associated with an increased risk of future CVD events for age, GFR, TAG and HDL-cholesterol [aRR (adjusted
among Type 2 diabetic individuals and, importantly, this relative risk), 1.55 (95 % CI, 1.23–1.95)] [43]. The
association was independent of classical risk factors, liver association between NAFLD and incident CKD was
enzymes and the metabolic syndrome. evident in the NAFLD group with elevated serum GGT
Studies have shown a link between NAFLD [aRR, 2.31 (95 % CI, 1.53–3.50)], even after adjustment
and increased carotid IMT (intima-media thickness). for age, GFR, TAG and HDL-cholesterol, but not
Fracanzani et al. [38] concluded (in a series of normal in the NAFLD group without elevated GGT [aRR,
and NAFLD subjects) that independent risk predictors 1.09 (95 % CI, 0.79–1.50); P = 0.008 for interaction)]
of increased IMT were the presence of hepatic steatosis [43].
(OR, 6.9), age (OR, 6.0) and increased systolic BP (OR,
2.3). More interestingly, Targher et al. [39] suggested that
the severity of liver histopathology among 85 NAFLD PATHOGENESIS
patients was strongly associated with early carotid
atherosclerosis, independent of age, gender, BMI, From the evidence presented above, it is important to
smoking, LDL-cholesterol, insulin resistance and the understand the pathogenesis of NASH because of the
presence of the metabolic syndrome. In addition, a large risk of progression to more severe end-stage liver disease.
population study has shown that NAFLD is associated It is also important to understand the relationship
with an increase in IMT [40]. On the other hand, in 100 between NASH and CVD. With respect to the latter, it is
dietcontrolled Type 2 diabetic individuals, the significant plausible that factors outwith the liver are contributing to
increase in carotid IMT in the presence of NAFLD has NASH and also to CVD. Changes in the liver associated
largely been explained by insulin resistance. Currently, with NASH may then compound the problem further
it is not clear how NAFLD leads to an increase in risk and amplify risk for CVD. For these reasons, we will
of CVD. One explanation may be the fact that NAFLD discuss the contribution of factors outwith the liver
appears to be associated with all metabolic risk factors to the pathogenesis of NAFLD and we will discuss
that are features of the metabolic syndrome. In addition, mechanisms within the liver contributing to NAFLD.
NAFLD may accelerate the progression of dyslipidaemia, There is a strong link between insulin resistance and
insulin resistance, atherosclerosis, endothelial dysfunc- excessive deposition of TAG in hepatocytes, which
tion, inflammation and oxidative stress. Interestingly, is the hallmark for diagnosis of NAFLD [44–46].
NAFLD may also be associated with a detrimental effect The excessive/ectopic fat depositions in the liver
on other organs that may have a direct or indirect influ- could be due to increased fatty acid delivery from
ence on CVD, or organs that may accelerate presentation adipose tissue, increased synthesis of fatty acid via
of CVD, at least in people with Type 2 diabetes. For the de novo pathway, increased dietary fat, decreased
example, NAFLD patients with Type 2 diabetes had mitochondrial β-oxidation, decreased clearance of
higher (P < 0.001) age- and gender-adjusted prevalence VLDL (very-LDL) particles or these factors in combin-
rates of both non-proliferative (39 compared with 34 %) ation. It is still a matter of debate whether insulin
and proliferative/laser-treated (11 compared with 5 %) resistance causes NAFLD or whether excessive accumul-
retinopathy, and CKD (chronic kidney disease) (15 ation of TAG, or precursors on the synthetic pathway,
compared with 9 %) than counterparts with Type 2 dia- precede and promote insulin resistance [47].
betes but without NAFLD. In these subjects, using
logistic regression analysis, NAFLD was shown to be Extrahepatic mechanisms contributing
associated with increased rates of CKD [OR, 1.87 (95 % to the pathogenesis of NAFLD
CI, 1.3–4.1); P = 0.020] and proliferative/laser-treated
retinopathy [OR, 1.75 (95 % CI, 1.1–3.7); P = 0.031] Lipolysis and NEFAs [non-esterified fatty acids
independently of age, gender, BMI, waist circumference, (‘free fatty acids’)]
hypertension, diabetes duration, HbA1c , lipids, smoking There is evidence that increased delivery of NEFAs to the
status and medication use [41]. Furthermore, in that liver from the peripheral (adipose) tissue is fundamental
study, NAFLD was associated with an increased incid- to the development of NAFLD. In support of this notion,
ence of CKD [42], independent of gender, age, BMI, waist studies in humans and rodents have shown that increased
circumference, BP, smoking, diabetes duration, HbA1c , NEFA delivery from adipose tissue is a significant source
lipids, baseline estimated GFR (glomerular filtration of fat accumulation in hepatocytes [48]. Some investigat-
rate), microalbuminuria and medication (hypoglycaemic, ors [49] have reported that approx. 60 % of fat deposited
lipid-lowering, antihypertensive or antiplatelet drugs). in hepatocytes is generated from adipose tissue sources.

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Metabolic disturbances in non-alcoholic fatty liver disease 545

Dietary macro- and micro-nutrients

Saturated fatty acids
The role of diet in the pathogenesis of NAFLD has
been investigated in humans and animal models [59,60].
Subjects on a high-fat diet develop fatty liver. Subjects on
a low-fat/high-carbohydrate diet also develop fatty liver
via increased de novo fatty acid synthesis [61]. In addition
to the above effect, high dietary fat intake is associated
with obesity and insulin resistance. As a consequence
of insulin resistance, there is impaired suppression of
lipolysis by insulin, leading to increased NEFA delivery
to the liver [62,63]. There is also reduced glucose uptake
in the fed state by adipose tissue and skeletal muscle,
resulting in hyperglycaemia and diversion of glucose to
the hepatic de novo pathway [64].
An increase in hepatic fatty acid is capable of exacerbat-
ing hepatic insulin resistance at the insulin receptor level
[65]. The mechanism has not been fully elucidated but is
thought to be due to translocation of the PKCδ (protein
kinase Cδ) isoform from the cytosol to the membrane
compartment, leading to impaired IRS (insulin receptor
substrate)/PI3K (phosphoinositide 3-kinase) activation
[55]. High dietary saturated fatty acids have been shown
to be associated with insulin resistance, NAFLD and
CVD [66]. In rats, high dietary saturated fatty acids
have been shown to promote ER (endoplasmic reticulum)
dysfunction and hepatocyte injury [67]. In another study
in humans [68], dietary consumption of saturated fat was
Figure 1 Interactions between liver, muscle and adipose reported to be higher in overweight patients with NASH
tissue in the control of VLDL secretion compared with age- and BMI-matched control subjects
(A) Normal state; (B) insulin-resistant state, showing an increased flux of NEFAs (14 compared with 10 % respectively).
from adipose tissue to the liver. FFA, NEFA; LPL, lipoprotein lipase.
n − 3 PUFAs (polyunsaturated fatty acids)
There is increasing interest in the role of n − 3 PUFAs in
In insulin-resistant subjects, there is a failure of insulin- NAFLD. The benefits of DHA (docosahexaenoic acid)
mediated suppression of HSL (hormone-sensitive lipase), and EPA (eicosapentaenoic acid) following the high con-
resulting in uncontrolled lipolysis in the adipose tissue sumption of fish oil have been shown in insulin-resistant
[48,50] (Figures 1A and 1B). HSL-knockout mice have animal models [66], including (i) decreased plasma NEFA,
decreased plasma NEFA and TAG concentrations, and TAG, glucose and insulin concentrations; (ii) decreased
have increased hepatic insulin sensitivity [51,52]. Peri- de novo lipogenesis, VLDL export and TAG con-
pheral (subcutaneous) fat constitutes a major proportion centrations; (iii) increased utilization and storage of gluc-
of the fat mass; however, it is not an absolute requirement ose by skeletal muscle; (iv) increased insulin-mediated
for developing fatty liver. Patients with lipodystrophy, glucose uptake by adipose tissue; and (v) decreased
who are also insulin-resistant with no peripheral and adipocyte size and visceral fat content. Other potential
intra-abdominal fat, develop fatty liver and severe hepatic benefits of PUFAs have also been reported in other animal
insulin resistance [53]. In this situation, the stimulus for experiments and these include: (i) decreased Kupffer cell
fatty liver may be increased NEFA flux to the liver activation [69]; (ii) decreased NF-κB (nuclear factor κB)
stimulating lipogenesis, combined with inadequate com- macrophage activation [70–72]; and (iii) decreased gene
pensatory fat oxidation, leading to fat accumulation over expression and production of inflammatory cytokines
time. Furthermore, there is some contribution to NEFA [70–73]. Similar findings have also been reported in hu-
flux to the liver from intra-abdominal fat in insulin- mans. In a small study of patients with NAFLD [74], there
resistant subjects [54]. Intra-abdominal fat is strongly was decreased TAG and glucose concentrations, liver
associated with reduced insulin sensitivity even in lean enzyme activities and hepatic steatosis following con-
subjects [55], and has been shown to correlate positively sumption of 1g of fish oil for 12 months. Another
with hepatic fat and hepatic insulin resistance [56–58]. study [75] showed similar results and decreased TNF-α

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546 C. D. Byrne and others

(tumour necrosis factor–α) concentrations after 2 g of

daily supplementation with fish oil for 6 months. The
authors [75] suggested that consumption of n − 3 PUFAs
could ameliorate NAFLD by improving steatosis,
inflammation and hepatocyte injury.
There is now some evidence that dietary n − 3 fatty
acids have a role in the pathogenesis of NAFLD and could
be a potential therapeutic target [76]. For example, there is
an association between PUFA deficiency and the develop-
ment of hepatic steatosis in animal models [66]. SREBP-
1c (sterol-regulatory-element binding-protein-1c) is a
key transcription factor for de novo lipogenesis (see
below) and is negatively regulated by n − 3 PUFAs [77].
Consequently, the activities of key enzymes for fatty acid
Figure 2 Potential control of dietary methyl donor
and TAG synthesis are down-regulated with decreased
regulation of gene expression relevant to NAFLD
hepatic fat deposition. PUFAs also negatively regulate
Dietary-independent pathway synthesis of PC is formed from SAM and PE in the
the activity of a glucose-responsive transcription factor
presence of PEMT, suggesting that a supply of methyl donors is important for the
[ChREBP (carbohydrate-responsive-element-binding
synthesis of PC.
protein); see below] by increasing ChREBP mRNA
decay and disrupting translocation of ChREBP from
the cytosol to the nucleus [78]. As a result of these effects,
it is plausible that high concentrations of dietary n − 3 has a distinct molecular composition with different
PUFA supplementation could be a promising treatment physiological functions dependent on the biochemical
for the management of NAFLD, and randomized pathway leading to PC generation [82,83]. PC from the
controlled trials in this area are urgently needed. dietary-independent pathway consists mainly of long-
chain PUFAs, whereas PC of dietary origin consists of
Carbohydrate and protein medium-chain saturated fatty acids [82]. PEMT activity
Excessive consumption of simple sugars has been has been shown to be important in the mobilization of
reported to promote the development of NAFLD. In essential fatty acids from liver [83].
one report, there was a 2–3-fold increase in de novo lipo- In animal models, PC generated from the PE pathway
genesis in both lean and obese subjects following a 4 day has been shown to be essential for the incorporation of
overfeeding with either a glucose or sucrose drink [79]. neutral lipids into VLDL particles [84], and for the normal
Similarly, high dietary intake of fructose also increases concentrations and structural composition of plasma
de novo lipogenesis in both animal models and humans VLDL [85,86]. Additionally, it has been shown that
[80,81]. PEMT-knockout mice have gender-specific differences
The precise role of dietary protein in the pathogenesis in the plasma concentrations of PC, cholesterol esters
of NAFLD is uncertain. There is a need for further and TAGs [85]. In one study [87], PEMT-knockout mice
investigation as whether there is a causal relationship were fed an MCD (methionine- and choline-deficient)
between excess dietary protein and NAFLD. diet resulting in significantly decreased hepatic PC
concentrations, severe liver damage and death. Animal
Micronutrients models (mice) that are fed an MCD diet develop NASH
The relationship between micronutrients and NAFLD is and this has provided a good model for studying the
now attracting considerable interest. PC (phosphatidyl- liver in NASH, although the mice tend to be thin and are
choline), the most abundant phospholipid in the mam- insulin-sensitive.
malian cell, is formed from dietary-dependent and Dietary deficiency of methionine in animal models
-independent pathways. In the presence of CCT is associated with reduced glutathione deficiency, with
(CTP:phosphocholine cytidylyltransferase) and CPT secondary free-radical-induced hepatocyte damage and
(CDP-choline:1,2 diacylglycerol choline phospho- inflammation [83]. As PC is essential for the synthesis of
transferase) enzymes, PC is formed from dietary choline VLDL particles, PC deficiency is associated with hepatic
and DAG (diacylglycerol). In the dietary-independent fat deposition secondary to the impaired export of VLDL
pathway, PC is formed from SAM (S-adenosylmethion- particles. PEMT-knockout mice had a significant increase
ine) and PE (phosphatidylethanolamine) in the presence in hepatic TAG and cholesterol ester concentrations [83],
of PEMT (phosphatidylethanolamine N-methyltrans- and there was almost a total reversal of these effects
ferase), suggesting that a supply of methyl donors following dietary choline supplementation, perhaps
is important for the synthesis of PC (Figure 2). There is suggesting a role for methyl donors in the generation of
some evidence that PC, from these different pathways, PC and NASH.

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Metabolic disturbances in non-alcoholic fatty liver disease 547

Intrahepatic mechanisms insulin sensitivity [94], suggesting that targeting ACC

activity could improve insulin resistance and ameliorate
De novo lipogenesis fatty liver.
Role of key enzymes FAS: FAS is the last key enzyme in de novo fatty
Under basal conditions, the contribution to hepatic acid synthesis and a total lack of FAS activity due to
fat from the de novo pathway is less than 5 % [88]; gene deletion is not compatible with intra-uterine life.
however, under pathological conditions, hepatic de novo Although this is a key lipogenic enzyme, FASKOL (liver-
lipogenesis also has been reported to be a significant specific FAS knockout) mice were also not protected
source of fat deposited in the liver (approx. 30 %) [47,48]. from developing hepatic steatosis. There was a 3-fold
In the de novo pathway, dietary glucose is converted into increase in malonyl-CoA, which would presumably
acetyl-CoA by L-PK (liver-specific pyruvate kinase). inhibit CPT-1 and decrease mitochondrial β-oxidation,
Acetyl-CoA is then converted into malonyl-CoA leading to increased hepatic NEFAs [95]. Increasing
and, subsequently, to fatty acids by actions of ACC liver fatty acids would presumably then lead to TAG
(acetyl-CoA carboxylase) and FAS (fatty acid synthase) synthesis and fat accumulation, worsening the NAFLD
respectively. phenotype, rather than ameliorating the phenotype.
ACC: The activity of ACC is regulated by the cell SCD-1 (stearoyl-CoA desaturase-1): MUFAs (mono-
energy status, insulin and the availability of NADPH. unsaturated fatty acids) are the major components of
ACC is inactivated by phosphorylation via AMPK TAGs, cholesterol esters and membrane phospholipids.
(AMP-activated kinase), resulting in decreased conver- The synthesis of MUFAs is catalysed by SCD-1.
sion of acetyl-CoA into malonyl-CoA, and PP2A Therefore modulating the activity of this enzyme may
(protein phosphatase 2A; an insulin-regulated enzyme) significantly decrease TAG synthesis. In a previous study
reverses this action by dephosphorylation of ACC. [96], SCD-1-knockout mice had decreased de novo lipo-
When cell energy is low with a high AMP/ATP ratio, genesis with increased mitochondrial fat oxidation. These
ACC is kept in its inactive phosphorylated form and, mice were protected from diet-induced obesity, fatty liver
consequently, acetyl-CoA is channelled to β-oxidation and insulin resistance when fed a high-fat and high-calorie
and ketogenesis for energy production. ACC is also diet. ASO inhibition of SCD-1 in liver and adipose tissue
under hormonal control by insulin and glucagon. Insulin also showed similar results with the SCD-1-knockout
stimulates PP2A, which dephosphorylates ACC thereby mice [97]. In another study [98], liver-specific SCD-1-
activating this enzyme and promoting lipogenesis. knockout mice were shown to have decreased gene
There are two isoforms of ACC namely ACC1 and expression for key lipogenic enzymes (ACC and FAS),
ACC2. ACC1, which is in the cytosol, is highly expressed with decreased de novo lipogenesis, and were also pro-
in liver and adipose tissue. ACC2, the mitochondrial tected from diet-induced hepatic steatosis. Additionally,
isoform, is expressed mainly in muscle and, to a lesser ex- the reduction in SCD-1 activity had a marked decrease
tent, liver. ACC2 is involved in the negative regulation of in the nuclear content of two key transcription factors
mitochondrial β-oxidation, hence increased β-oxidation for lipogenesis SREBP-1c and ChREBP (see text below).
seen in ACC2-knockout animals. This is due to decreased
malonyl-CoA, which derepresses CPT-1 (carnitine DGAT-2 (diacylglycerol acyltransferase-2): There is
palmitoyltransferase-1) activity [89]. Fatty acid synthesis now increasing interest in the role of DGAT-2, the
takes place in the cytosol and, therefore, only the ACC1 enzyme that catalyses the final step in TAG synthesis.
isoform is important in the de novo pathway for fatty Theoretically, inhibition of DGAT-2 should prevent the
acid synthesis. This occurs because there is no access to synthesis of TAG and NAFLD, although this appears
malonyl-CoA produced in the mitochondria by ACC2 not completely true as, in a recent experiment in db/db
[90,91]. In a LACC1KO (liver-specific ACC1-knockout) mice [99], inhibition of DGAT-2 with ASOs produced
model, Mao et al. [92] showed a 70 % reduction in hepatic surprising results. Feeding mice an MCD diet caused the
malonyl-CoA relative to control, a 50 % reduction in development of a fatty liver after 4 weeks. Although there
de novo lipogenesis and a 40 % decrease in TAG was marked improvement in hepatic steatosis following
concentrations. Paradoxically, this mouse model was inhibition of DGAT-2 with ASO, paradoxically, there
not protected from high-fat high-calorie diet-induced was worsening/progressive inflammation, hepatocyte
obesity, fatty liver and insulin resistance, despite a injury and fibrosis in these mice. Histological sections
significant decrease in fat deposition. This effect may of the liver in the DGAT-2 arm revealed marked inflam-
be due to increased compensatory activity of liver- matory changes and hepatocyte necrosis. Additionally,
specific ACC2 [93]. In contrast, in vitro ASO (antisense there was worsening liver fibrosis with raised mRNA
oligonucleotide) inhibition of both ACC1 and ACC2 led for most of the markers of fibrosis and hepatic stellate
to a significant decrease in hepatic malonyl-CoA concen- cell activation. Although an MCD diet has been
trations, decreased hepatic lipids and increased hepatic shown to increase the expression of fibrosis markers

C The Authors Journal compilation 
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548 C. D. Byrne and others

Figure 3 Effect of dietary fatty acid intake and de novo fatty acid synthesis in regulating lipid synthesis
Potential interactions are shown between hepatic lipid metabolism, DNA methylation and gene expression, inflammation, VLDL secretion and hepatic triacylglycerol
accumulation in the pathogenesis of NAFLD. FACoA, fatty acyl-CoA; UCP-2, uncoupling protein-2.

[TGF-β 1 (transforming growth factor-β 1 ), α-SMA of TAG. Further research in this area is clearly needed
(α-smooth muscle actin), TIMP-1 (tissue inhibitor to elucidate which molecule(s) on the TAG synthetic
of metalloproteinases-1) and collagen], there was a pathway are able to trigger inflammatory pathways.
failure of inhibition of most of these markers after
ASO inhibition of DGAT-2. The plausible explanation Role of key transcription factors
for the progressive inflammation and fibrosis may SREBP-1c: Evidence is now emerging that SREBP-
be due to increased hepatic NEFAs and the gene- 1c is a positive transcription factor for ACC and FAS
ration of hepatotoxic free radicals [99]. Mice in the genes [100], and we have suggested that abnormalities in
treatment arm had significantly higher hepatic fatty acids. SREBP-1c function may play an important pathogenetic
Although an MCD diet inhibits mitochondrial fat oxid- role in contributing to the NAFLD phenotype (see
ation, there was amplification of this effect in the Figure 3) [101]. Overexpression of SREBP-1c in adult
ASO-inhibition mice with a diversion of fatty acids rats causes a 26-fold increase in fatty acid synthesis and
to microsomal and peroxisomal fat oxidation. Conse- fat deposition. Apart from modulating the key lipogenic
quently, in response to increased microsomal fat oxid- enzymes (ACC and FAS), SREBP-1c also stimulates gene
ation, there was increased microsomal enzyme [Cyp2E1 expression for fatty acid elongation and TAG synthesis.
cytochrome P450 2E1)] activity with increased (micro- However, SREBP-1c is by itself insufficient for full tran-
somal) production of ROS (reactive oxygen species). scription activity of fatty acid synthesis. In a knockout
Interestingly, there was improved systemic insulin experiment, only a 50 % reduction in fatty acid synthesis
resistance after ASO inhibition of DGAT-2. There was a was reported following SREBP-1c knockout [102].
reduction in weight, and reduced serum NEFAs, glucose Current evidence indicates that the effect of insulin on
and insulin concentrations. There was also reduced lipogenesis is mediated via SREBP-1c activity [103,104].
TNF-α and increased serum adiponectin concentrations. In an insulin-resistance state with hyperinsulinaemia,
In view of this evidence, the authors [99] postulated that the transcriptional activity of SREBP-1c is up-regulated
TAG itself may not be harmful, but could instead protect and both insulin and SREBP-1c synergistically stimulate
the liver from lipid toxicity and hepatotoxic free-radical key lipogenic genes, thereby promoting de novo
damage by buffering hepatic fatty acids into the synthesis fatty acid synthesis. Insulin-resistant ob/ob mice have

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Metabolic disturbances in non-alcoholic fatty liver disease 549

increased concentrations of SREBP-1c and also develop SHIP-2 is an insulin-signal-regulatory phosphatase,

spontaneous fatty liver. Increased SREPB-1c in these but the absence or reduced expression and activity of
mice increases lipogenic gene expression, increases fatty this enzyme has not been reported to be associated with
acid deposition and accelerates TAG deposition in fatty liver [112–114].
hepatocytes [105,106]. Administration of leptin, which PTEN is a tumour-suppressor protein, but with phos-
opposes the action of SREBP-1c, reverses these metabolic phatase activity, and has a regulatory effect on the insulin
derangements [107]. In contrast, in the fasting state, when signalling pathways [111,114,115]. The substrate proteins
insulin production is low but glucagon concentrations are for this enzyme are PtdIns(3,4)P2 and PtdIns(3,4,5)P3 .
increased, the quantity of SREBP-1c in liver is decreased, As a phosphatase, the physiological function of PTEN
although this effect is reversed by re-feeding [108,109]. is to dephosphorylate the second messengers generated
by the activation of PI3K, thereby down-regulating or
Forkhead transcription factors: Decreased mitochon-
terminating insulin signalling downstream of PI3K [111].
drial fatty acid oxidation is also contributory to excessive
Overexpression of PTEN has been shown to have in-
fat deposition in the liver in insulin-resistant subjects.
hibitory effects on insulin signalling, including decreased
A plausible mechanism for this effect could be mediated
Akt activation and GLUT4 (glucose transporter 4) trans-
via phosphorylation and dephosphorylation of the
location to the cell membrane [116,117]. Overexpression
transcription factor Foxa2 (forkhead box a2) [110].
of PTEN in muscle from obese fa/fa Zucker rats had been
Under physiological conditions, active (dephos-
shown to contribute to muscle insulin resistance in these
phorylated) Foxa2 promotes mitochondrial fatty acid
animals [118]. In contrast, down-regulation of PTEN has
oxidation, whereas insulin reverses this effect via phos-
the opposite effect, with increased glucose uptake in fat
phorylation by IRS-1 and IRS-2. Paradoxically, in
and muscle in response to insulin [119]. In mice, liver-
insulin-resistance, Foxa2 is still predominantly in its
specific deletion of PTEN has been shown to increase
inactive form because of residual sensitivity to insulin in
insulin sensitivity, but paradoxically causes fatty liver
the liver resulting in decreased β-oxidation and increased
disease and hepatocellular cancer [120,121]. The mech-
hepatic fat deposition.
anism for this paradox is yet to be clarified, but there has
INSIG-1 (insulin induced gene-1), insulin signalling, been a number of hypotheses suggested, some of which
PTEN (phosphatase and tensin homologue deleted on highlight the lack of negative regulation on the insulin
chromosome 10) and mTOR (mammalian target of rapa- signalling pathways by PTEN. In PTEN-knockout mice,
mycin): There is now some interest on the role of it has been shown that there was increased synthesis and
INSIG-1 and fatty liver. INSIG -1 is highly expressed in storage of TAG in hepatocytes due to the up-regulation
hepatocytes and adipocytes, and has been suggested to of PI3K/Akt activity [119,121]. As a consequence of the
act as the ‘brake’ for lipogenesis in fat and limiting TAG lack of PTEN activity, there is increased hepatocyte fatty
deposition in hepatocytes [110a]. The gene may also acid uptake, increased fatty acid synthesis and increased
play a modulatory role in preadipocyte diffentiation. It esterification of fatty acids to TAG. Taken together, these
is possible that the benefits of PPAR-γ (peroxisome- findings suggest that decreased PTEN activity would lead
proliferator-activated receptor-γ ) agonists in NASH are to excessive fat deposition in the liver.
due to the regulation of INSIG-1 by these drugs. There is evidence that reduced or absent expression
Under physiological circumstances, the phosphoryl- of liver-specific PTEN is associated with hepatic
ation of IRS, PI3K, Akt and the downstream proteins steatosis, inflammation, fibrosis and neoplasm [115]. In
are essential for the action of insulin in insulin-sensitive one study, PTEN-deficient mice were shown to have
organs. However uncontrolled phosphorylation would biochemical and histological evidence, including fibrosis
lead to amplification of insulin action with insulin hyper- of NASH, present at 40 weeks age [115]. The mechanism
sensitivity, whereas a lack of phosphorylation of the for NASH in this animal model has been reported to be
insulin substrate proteins could cause insulin resistance. due to increased expression of the PPAR-γ , SREBP-1c
There are key negative regulatory proteins (phosphatases) and downstream genes, including Akt and Foxo1,
that physiologically terminate the action of insulin by resulting in increased lipogenesis, inflammation and
dephosphorylation, and constitutive action of these fibrosis [115]. The reason for the increase in fat deposi-
proteins could lead to decreased insulin sensitivity. tion in the liver could be partly due to the increased
Other insulin signalling counter-regulatory mechanisms expression of SREBP-1c. As SREBP-1c is a key
include: (i) serine or threonine phosphorylation of the transcription factor for lipogenesis with increased ACC,
insulin receptor; (ii) activation of tyrosine phosphatases; FAS and SCD-1 enzyme activities, all of these act syner-
(iii) inhibition of insulin receptor and IRSs by the SOCS gistically to promote fatty acid synthesis.
(suppressor of cytokine signalling) family; and (iv) activ- As a consequence of increased PPAR-γ expression,
ation of phosphoinositide phosphatases [PTEN, SHIP-2 there is also a secondary induction of key enzymes
(SH2-containing inositol phosphatase-2) and myotubu- involved in mitochondrial β-oxidation. As a result,
larin] [111]. the increase in fat oxidation with a marked increase in the

C The Authors Journal compilation 
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550 C. D. Byrne and others

Figure 4 Interaction between NEFAs, mTOR and NF-κB

Schematic diagram showing the effects of increased availability of fatty acids in
Figure 5 Regulation of the mTOR signalling pathway by
hepatocytes resulting in activation of mTOR. The activation of mTOR leads to
nutrients and insulin
activation, dissociation and then translocation of NF-κB from the cytosol to the
Schematic diagram showing the up-regulation of mTOR by insulin and
nucleus, where it regulates PTEN at the level of transcription. As a consequence,
nutrient-sensing mechanisms and the down-regulation of mTOR by AMPK activation.
there is amplification of insulin signalling with increased hepatic lipogenesis.
∗ Activation of mTOR leads to activation of the downstream proteins including S6
Indicates activation of the protein. FFA, NEFA.
ribosomal protein resulting in serine phosphorylation of IRS-1 and consequent
decreased insulin action.
generation of oxidative free radicals leads to inflammation
and fibrosis via activation of the NF-κB pathway
[122,123]. For example, there is a 7-fold increase in the protein)-binding protein)]/p300, the transcriptional
hepatic concentration of H2 O2 in PTEN-deficient mice activator for PTEN [124].
compared with the wild type [115]. There is also evidence mTOR is a downstream target of the PI3K signalling
of transcriptional down-regulation of PTEN in the liver pathway, but the activation of this protein down-regulates
with decreased enzyme protein levels in fatty liver disease, insulin signalling in insulin-responsive tissue [125,126].
and this has been shown in the liver of obese insulin- The mechanism is through serine phosphorylation of
resistant ZDF (Zucker diabetic fatty) rats with diabetes IRS-1 [127]. The regulation of the mTOR signalling path-
and hepatic steatosis [114]. This raises the question of way is complex. This pathway is activated by hormonal-
cause or effect. Furthermore, only unsaturated NEFAs and nutrient-sensing factors, whereas inhibitory regul-
have been shown to decrease the expression of PTEN ation is through AMPK activation. Insulin via IRS
in hepatocytes, suggesting that this is a specific effect activates PI3K, which, in turn, activates Akt. Akt then ac-
of unsaturated fatty acids. For example, the down- tivates mTOR via phosphorylation. The activation of
regulation of PTEN expression by the unsaturated fatty mTOR leads to phosphorylation and activation of down-
acid oleic acid is via the activation of mTOR and/or stream proteins, including p70S6 kinase and S6 ribosomal
NF-κB pathways. Similar findings have been reported protein. As a consequence of S6 ribosomal protein phos-
in humans in a small study of morbidly obese subjects phorylation, serine phosphorylation of IRS-1 occurs at
(n = 20), where there was a down-regulation of PTEN ex- Ser636 /Ser639 [128]. Because of the serine phosphorylation
pression with a biopsy-proven decrease in immunohisto- of IRS-1, this key insulin substrate protein becomes un-
chemistry staining for PTEN in subjects with hepatocytes responsive to insulin signals and, instead, undergoes rapid
steatosis of > 80 % [114]. degradation and there is a decreased interaction between
In insulin-resistant subjects, there is an increase the serine-phosphorylated IRS-1, the insulin receptor
in plasma NEFA concentrations with an increase in (upstream) and PI3K [129], resulting in decreased insulin
delivery and uptake of NEFAs by hepatocytes. This action (Figure 5).
results in the activation of mTOR, which in turn leads to The mTOR signalling pathway is also regulated via
activation of NF-κB [114]. As both mTOR and NF-κB a nutrient-sensing mechanism. An increase in plasma
exist as a complex, there is a subsequent dissociation and leucine concentrations is a potent activator of the mTOR
then translocation of NF-κB into the nucleus, where it pathway through class III PI3K activation (class I PI3K
down-regulates the expression of PTEN at the level of being the substrate protein of the insulin/IRS pathway)
transcription (Figure 4). The precise mechanism of how [130]. A high-fat diet is associated with obesity, hepatic
NF-κB transcriptionally down-regulates PTEN is not fat deposition, hepatic and whole-body insulin resistance
fully understood, but could be through the sequestration and, in an animal experiment, the mTOR signalling
of the CBP [CREB (cAMP-response-element-binding pathway has been shown to be up-regulated in mice fed

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Metabolic disturbances in non-alcoholic fatty liver disease 551

a high-fat diet compared with controls [131]. As insulin serine instead of tyrosine phosphorylation and insulin
activates the mTOR pathway, we can assume that up- resistance [138,140]. ROS and TNF-α are also capable of
regulation of this pathway may be due to increased fasting activating JNK. The overall effect is decreased glucose
insulin concentrations associated with high dietary fat uptake with persistent hyperglycaemia without or with
and insulin resistance. Under normal conditions, this hyperinsulinaemia. This will exacerbate de novo hepatic
action of insulin would be mediated via tyrosine lipid synthesis via activation of SREBP and ChREBP
phosphorylation of the insulin receptor; however, in this being driven by insulin and glucose respectively.
animal experiment, there was no difference in tyrosine
phosphorylation of the insulin receptor or the down- LXR-α (liver X receptor-α): ChREBP is also regulated
stream target proteins in high-fat-fed mice and the control by LXR-α, and important ligands for LXR-α are oxy-
group. The authors [131] hypothesized that the activation sterol, insulin and glucose. LXR-α is a member of the
of the mTOR pathway was through a poorly understood nuclear receptor family that plays an important role in
insulin/IRS-independent pathway, probably due to acute lipogenesis. LXR-α exerts transcriptional control on
fat deposition in the liver. However, in the same report SREBP-1c and, therefore, indirectly on ACC and
[131], the investigators were not able to show that the FAS [141–143]. ob/ob mice that are insulin-resistant
up-regulation of the mTOR pathway was due to acute develop fatty liver, although this is unlikely to be due to
hepatic steatosis. Pharmacological induction of acute hep- insulin-mediated effects on ACC and FAS. The explan-
atic steatosis in mice showed a 5-fold increase in hepatic ation for this paradox may be that not all insulin-sensitive
TAG content, but failed to show a significant increase in metabolic pathways are uniformly affected by insulin
the activation of mTOR and the downstream proteins, resistance. A plausible mechanism is via glucose control.
leading to the authors concluding that acute hepatic The genes for the gluconeogenic enzymes PEPCK (phos-
steatosis is, by itself, insufficient for sustained activation phoenolpyruvate carboxykinase) and G6Ptase (glucose-
of the mTOR signalling pathway [131]. 6-phosphate translocase) are up-regulated in the liver of
Increased fatty acid delivery has been shown to up- insulin-resistant ob/ob mice [144]. High hepatic glucose
regulate the mTOR pathway, thereby exacerbating hep- output is a consequence of increased gluconeogenesis.
atic insulin resistance. In a recent study [132], in the ab- We may then postulate that the enhanced transcriptional
sence of insulin, palmitic-acid-cultured hepatocytes had activities of the glycolytic and lipogenic genes are regu-
activation of the mTOR pathway with a 4-fold increase lated by the co-ordinated activity of ChREBP, LXR-α
in p70S6 kinase activity. In the presence of insulin, there and SREBP-1c.
was an amplification of this effect. The antidiabetic agent
metformin, via activation of AMPK, was shown to reverse Inflammation, oxidative stress, fibrosis
this effect. In insulin-resistant subjects with increased and ER stress
fatty acid delivery to the liver and hyperinsulinaemia, Fat is now considered a metabolically active endocrine
we postulate that worsening insulin resistance may be organ producing pro-inflammatory cytokines, including
due to sustained or enhanced activation of the mTOR TNF-α, IL (interleukin)-6 and IL-8, and there is evi-
pathway. dence to support the activation of other inflammatory
pathways, oxidative stress and the de novo pathway by
ChREBP: The gene for l-pyruvate kinase, a key com- TNF-α [145–147]. It has recently been hypothesized
ponent of the de novo pathway for fatty acid synthesis, that NAFLD is a consequence of a ‘two hit’ insult. As
is not regulated by SREBP-1c [133], but exclusively by a consequence of insulin resistance, there is excessive
glucose [134]. accumulation of TAG in hepatocytes, which is the
A glucose-responsive transcription factor, ChREBP, ‘first hit’. Oxidative stress from β-oxidation, increased
has been described and thus illustrates how glucose expression of inflammatory cytokines by NF-κB-
affects gene transcription [135]. ChREBP is controlled dependent pathways and adipocytokines are potential
by glucose and regulates the translocation of this protein synergistic factors acting in concert as the ‘second hit’,
from cytosol to the nucleus [136]. In the nucleus, glucose resulting in hepatocyte injury, inflammation and fibrosis.
promotes the binding of this protein to the carbohyd- In contrast, all subjects with NAFLD have insulin
rate-response element in the promoter region of target resistance, even in lean subjects and those with normal
genes [137]. glucose tolerance [59,148,149]. The core metabolic
As part of deranged insulin signalling in obese insulin- derangement is due to insulin resistance resulting
resistant subjects, there is decreased GLUT4 transloca- in increased lipolysis in adipose tissue, increased
tion to the cell membrane in muscle and adipose tissue. NEFA uptake by hepatocytes and increased TAG
The investigators suggested that fatty acyl-CoA and other synthesis in the liver. Mitochondrial fat oxidation and
NEFA derivatives and TNF-α impair the activation of export of VLDL particles are not able to match TAG
IRS and PI3K [138,139]. Consequently, there is activation synthesis, leading to net fat deposition in the hepato-
of JNK (c-Jun N-terminal kinase) pathways, leading to cytes. As a consequence of abnormal fat accumulation

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552 C. D. Byrne and others

in the hepatocytes, there is marked derangement in the However, it is uncertain whether inflammatory
insulin signalling pathways in the liver via activation of cytokines cause NAFLD or occur as a consequence of
NF-κB, IKK-β [IκB (inhibitor of NF-κB) kinase-β], the developing liver condition and NF-κB activation. In
atypical PKC and JNK pathways by NEFAs [150,151]. a previous review [166], increased inflammatory cytokine
There is evidence that increased mitochondrial fat production was reasoned to be a consequence of IKK-β
oxidation, a consequence of hepatic insulin resistance, is and NF-κB activation and, in support of this reasoning
linked to hepatic injury, inflammation and fibrosis. This in NAFLD, increased hepatic NF-κB activity in high-
is because NEFAs and their metabolites are ligands for fat-fed mice was associated with increased inflammatory
PPAR-α. This transcription factor regulates a number cytokine expression, with attenuation of this effect with
of genes, including those involved in mitochondrial, specific inhibition of NF-κB activity [150]. Additionally,
peroxisomal and microsomal fat oxidation, and there is activation of the upstream protein IKK-β was also
now evidence to support the up-regulation of these genes associated with inflammation, hepatic and systemic insu-
in NAFLD. lin resistance, and there was also a reversal of this effect
As a result of increased fat acid oxidation in the using a hepatic-specific inhibitor of NF-κB. In NAFLD,
mitochondria and peroxisome, there is increased it is plausible that hepatic NEFAs have a toxic effect on
generation of hepatotoxic oxygen free radicals. Because hepatocyte lysosomal membranes with the consequent
of oxidative stress, there is lipid peroxidation and severe release of proteolytic enzymes that directly activate the
mitochondrial dysfunction with structural and functional IKK-β and NF-κB pathways with increased TNF-α
abnormalities. There is ATP depletion due to the failure expression [167].
to generate ATP via oxidative phosphorylation [152,153], JNK is a member of the MAPK (mitogen-activated
and this could be due to increased expression of UCP-2 protein kinase) family that regulates cell function via the
(uncoupling protein-2) by PPAR-α activation [154,155]. control of AP-1 (activated protein-1), and there is a strong
Patients with NASH also have structural mitochondrial association between increased JNK activity and insulin
abnormalities [156–160], and, under-expression of some resistance. In obesity with insulin resistance, increased
genes necessary for normal mitochondrial function have NEFAs and inflammatory cytokines are accompanied
also been reported in these patients [161]. by increased JNK activity in insulin-sensitive tissue,
Inflammation is the link between obesity and insulin thereby causing insulin resistance [162]. Although a
resistance, and may have an important role in the knockout model for JNK1 did not develop insulin
pathogenesis of hepatic and systemic insulin resistance. resistance [168], a possible role for the JNK family of
For example, serine phosphorylation of IRS-1 by inflam- MAPKs in NAFLD is presently uncertain.
matory cytokines is a mechanism mediating insulin The ER is a cellular organelle for synthesis, storage and
resistance in obese subjects with chronic low-grade transport of proteins. Because of the presence of some
inflammation. In support of an association between chaperone proteins, the ER ensures proper folding of pro-
insulin resistance and inflammatory cytokines, there is teins. IRE-1 (inositol-requiring enzyme-1), PERK [PKR
increased production of TNF-α in obese animal models (double-stranded-RNA-dependent protein kinase)-like
and in obese insulin-resistant subjects [162]. In contrast, ER kinase), ATF-6 (activating factor-6) and XBP-1
knockout mice for either TNF-α or the TNF-α receptor (X-box-binding protein-1) are subcellular proteins
were reported to be insulin-sensitive [162]. In obese necessary for ER function. Although these protein are
subjects with previously increased TNF-α expression, important for normal ER function, abnormal activation
decreased TNF-α expression following weight loss and may occur as a consequence of a ‘stressed’ environment,
exercise was observed [163]. The molecular mechanism for example hypoxia, leading to inflammation and insulin
responsible for TNF-α-induced insulin resistance is resistance [169]. Evidence is beginning to emerge for the
most probably serine phosphorylation of IRS-1, which pathogenic role of hepatic ER ‘stress’ in inflammation
has an inhibitory effect on the downstream propagation and insulin resistance. For example, ER ‘stress’ has been
of insulin signals [163,164]. shown to directly activate the IKK-β/NF-κB and the
Another mechanism for TNF-α-induced insulin JNK/AP-1 pathways via interactions with IRE-1 and
resistance is through the activation of the IKK-β PERK, thereby inducing or exacerbating inflammation
pathway [165]. Overexpression of IKK-β is associated and insulin resistance [162,166] (Figure 6). Hepatic ER
with reduced insulin signalling in cultured cells [165]. ‘stress’ has also been shown to occur with high-fat
In contrast, liver-specific deletion of IKK-β resulted in feeding and in a genetically induced obese animal model
improved hepatic insulin sensitivity even on a high-fat [170].
diet [165]. As a result of the activation of IKK-β, there Because of the modulatory effect of endogenous bile
is downstream activation of the NF-κB pathway with acids and their derivatives on ER function, the therapeutic
worsening inflammation and hepatic insulin resistance. potential of these pharmacological agents have been
At the level of transcription, the activation of NF-κB explored in both humans and animal models. Treatment
also has an inhibitory effect on PPAR-γ agonism. of NASH in insulin-resistant subjects with UDCA

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Metabolic disturbances in non-alcoholic fatty liver disease 553

κB activation following injection of LPS in the liver

of NEMO-knockout mice, and the administration of
TNF-α in these mice also showed a similar effect [171].
As a consequence of the absence of NF-κB activation,
these mice developed severe liver failure due to exposure
to LPS and increased cytokine drive. Thus LPS-induced
activation of TNF-α could contribute to liver damage
in the pathogenesis of NAFLD, particularly if NEMO-
dependent NF-κB activation is inadequate to protect
the liver from damage exacerbated through activation
of the JNK pathway. It is possible that oxidative stress
may be a contributory factor to hepatocyte necrosis
observed in NEMO-knockout mice. In an animal
study [171], NEMO-knockout mice were treated with
Figure 6 Interaction between inflammatory cytokines, ER
a dietary antioxidant supplement, BHA (butylated
stress, JNK, NF-κB pathways and hepatic inflammation
hydroxyanisole), and were shown to have biochemical
Schematic diagram showing activation of JNK/AP-1 and IKK-β/NF-κB pathways
and histological improvements in chronic liver disease.
by ER stress and inflammatory cytokines resulting in exacerbated inflammation.
There was also reduced hepatic JNK activity in the
antioxidant group, leading the authors to postulate
(ursodeoxycholic acid) has so far been disappointing that antioxidant therapy could ameliorate or normalize
[168], although, in obese mice, these authors have shown chronic liver disease in this NEMO-knockout model.
improved hepatic and whole-body insulin sensitivity
with UDCA treatment.
There is emerging interest in the role of adipocytokines
NEMO (NF-κB essential modulator/IKK-γ ) is the
in the pathogenesis of NAFLD. Adiponectin has been
regulatory component of the IκB complex. The two cata-
shown to decrease de novo fatty acid synthesis but en-
lytic components of this complex are IKK-1/IKK-α and
hance fat oxidation. Stimulation of adiponectin receptors,
IKK-2/IKK-β [171]. NF-κB has an important cellular
which are expressed in hepatocytes and myocytes, leads
function in the regulation of immune and inflammatory
to PPAR-α and AMPK activation [175]. As a result,
responses, and is maintained in its inactive form in the
adiponectin increases β-oxidation but decreases TAG
cytosol by binding inhibitory proteins [171]. Although
synthesis. Adiponectin also has direct anti-inflammatory
the physiological function of NF-κB in adult liver is
effects by decreasing hepatic TNF-α production. In
not known, deletion of either the catalytic component
support of a pathogenetic role for adiponectin in affecting
(IKK-2) or the regulatory component (NEMO) is not
inflammation in NAFLD, patients with NASH have
compatible with intra-uterine life [172].
decreased expression of adiponectin receptors in the
Hepatocellular carcinoma is a well-known complic-
liver and decreased serum adiponectin concentrations
ation of NAFLD and the molecular mechanism in this
compared with patients with simple steatosis [176,177].
group of patients in beginning to be better understood.
There is evidence to support the tumour-suppressive Endocannabinoids
effect of NEMO in hepatocytes. In one animal The psychotropic actions of the plant Cannabis sativa
experiment [171], NEMO-knockout mice developed were first documented approx. 4000 years ago. The
spontaneous hepatocellular carcinoma at 12 months of endocannabinoid system contributes to the physiological
life. In addition, there was histological evidence of pre- regulation of energy balance, food intake, and lipid and
malignant lesions in the liver of these mice by 9 months. glucose metabolism through both central and peripheral
In the same study, there was biochemical and histological effects. Cannabinoid receptors, named CB1 receptor and
evidence of NASH prior to the development of cancer CB2 receptor, participate in the physiological modulation
in this mouse model. In contrast, specific deletion of of many central and peripheral functions. The CB2
IKK-2 (a catalytic component) was not associated with receptor is mainly expressed in immune cells, whereas the
chronic liver disease or spontaneous liver cancer [171]. CB1 receptor is the most abundant G-protein-coupled
These findings led the authors to suggest that NEMO- receptor expressed in the brain. The CB1 receptor is
dependent NF-κB activation has a physiological role in expressed in the hypothalamus and the pituitary gland,
the prevention of spontaneous hepatic cancer. and its activation is known to modulate all the endocrine–
LPS (lipopolysaccharide) is a potent inducing agent of hypothalamic–peripheral endocrine axes. Recent exciting
TNF-α and other inflammatory cytokines [173,174], and new results suggest that inhibition of the endocannabin-
bacterial antigens entering between tight junctions in the oid system might be beneficial in the treatment of the
intestine could have an impact on liver damage in NAFLD. Interestingly, a new class of anti-obesity med-
the pathogenesis of NAFLD. There is a failure in NF- ication, such as rimonbant (a CB1 receptor antagonist),

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554 C. D. Byrne and others

was shown to be beneficial in an animal model of hepatic [TNF-α, TGF-α, IL-6 and IL-8] via the activation of
steatosis [178]. Treatment of obese (fa/fa) rats with NF-κB [186]. NEFAs accumulate in the hepatocytes in
rimonabant (30 mg/kg of body weight) daily for 8 weeks NAFLD and are also capable of activating the NF-κB
abolished hepatic steatosis, decreased hepatomegaly, pro-inflammatory pathway [166,187].
decreased ALT and GGT levels, decreased dyslipidaemia In addition to the NF-κB-dependent pathways,
and, importantly, increased the level of the anti- Kupffer cells (liver-specific activated macrophages) are a
inflammatory hormone adiponectin [178]. At the hepatic source of inflammatory cytokines and, in an obese indi-
cell level, activation of hepatic CB1 receptors by stellate vidual, adipose tissue with infiltrating macrophages is also
cell endocannabinoid 2-AG (2-arachidonoylglycerol) is a source of pro-inflammatory cytokines. Because of the
responsible for ethanol-induced steatosis by increasing increased production of pro-inflammatory cytokines in
lipogenesis and decreasing fatty acid oxidation [179]. NAFLD, these cytokines (e.g. TNF-α) could exacerbate
Recently, rimonabant has been shown to be effective in systemic and hepatic insulin resistance with worsening
reducing weight [180], and randomized clinical trials are inflammation and fibrosis. Inflammatory cytokines
now needed to establish whether rimonabant produces cause impaired insulin signalling, cell damage, neutrophil
therapeutic benefit in the treatment of NAFLD. chemotaxis, hepatic stellate cell activation and apoptosis
[188–190], and there is increased expression of TNF-α
Mitochondrial dysfunction and ROS and its receptor in liver and adipose tissue in obese
The mitochondrial genome synthesizes 13 respiratory individuals with NASH, which correlates with the
chain polypeptides and is very sensitive to oxidative degree of fibrosis [191].
damage, probably because of (i) the proximity of mtDNA Under physiological conditions, ketogenesis is ex-
(mitochondrial DNA) to the inner mitochondrial quisitely sensitive to insulin but, paradoxically, mito-
membrane (a source of ROS), (ii) the absence of chondrial fat oxidation and ketogenesis are increased
protective histones; and (iii) the lack of effective DNA in NAFLD with hyperinsulinaemia [192,193]. Even
repair mechanisms. obese leptin (ob/ob)-deficient mice with fatty liver also
There is evidence that increased production of ROS, have increased fat oxidation [194], perhaps explained
pro-inflammatory cytokines and hepatocyte necrosis by increased concentrations of long-chain fatty acids
secondary to mitochondrial dysfunction is important activating PPAR-α, a nuclear receptor and a positive
in the pathogenesis of NAFLD. Although the sequence transcription factor for the CPT-1, MCAD (medium-
of pathophysiological events are yet to be clarified, it is chain acyl-CoA dehydrogenase) and HMG (3-hydroxy-
plausible that oxidative stress is an early event in NAFLD 3-methylglutaryl)-CoA synthetase genes. Some investig-
and could provide the ‘trigger’ for inflammation and ators have postulated that long-chain fatty-acid-mediated
fibrosis. Oxidative stress is undoubtedly a major feature PPAR-α activation is specific to fatty acids from the
of NASH, and potential sources of ROS are hepatocyte de novo pathway [95], and there is increased expression
parenchymal cells, chronic inflammatory cells within the of PPAR-α in ob/ob mice with steatosis [195]. Long-
hepatocytes and, in obese subjects, adipose tissue with chain fatty acids increase the activity and expression of
infiltrating activated macrophages [181,182]. CPT-1 and, thereby, increase fat oxidation [196]. Addi-
There is some evidence that increased generation of tionally, there is a decreased affinity of CPT-1 activity for
ROS could enhance progression from simple steatosis malonyl-CoA, a potent inhibitor of CPT-1 [197].
to steatohepatitis and fibrosis [183]. The potential Activation of AMPK also enhances mitochondrial fat
mechanisms are through lipid peroxidation, induction oxidation via phosphorylation (inactivation) of ACC.
of pro-inflammatory cytokines and induction of FAS As a result, there is a reduced conversion of acetyl-CoA
ligand. The plasma and mitochondrial membranes are into malonyl-CoA and, therefore, decreased inhibition
vulnerable to free radical damage, and ROS-induced of CPT-1 by malonyl-CoA, resulting in increased
lipid peroxidation of membranes can cause cell necrosis β-oxidation [198].
or apoptosis. Lipid peroxidation can also cause immuno- In mitochondria oxidative phosphorylation, NADH
logical dysfunction, which could lead to hepatic fibro- and FADH2 are re-oxidized to NAD+ and FAD via
genesis. It is possible that, as a result of lipid peroxid- the electron transport chain. As part of oxidative phos-
ation, there is release of MDA (malondialdehyde) and phorylation, this is a co-ordinated flow of electrons down
4-HNE (4-hydroxynonenal), which bind hepatocyte an electrochemical gradient from complex I to IV with
proteins forming new antigen(s) and, thereby, provoking simultaneous extrusion of protons, creating a large elec-
a harmful immunological response. An immunological trochemical gradient. Consequently, a large gradient
response could induce neutrophil chemotaxis and activate across the mitochondrial membrane drives ATPase syn-
hepatic stellate cells to promote collagen synthesis and thase resulting in ATP production. Under physiological
deposition [184,185]. conditions, most of the electrons provided to the
Oxidative stress (ROS) has also been shown to respiratory chain ‘transit’ through to the last complex,
increase production of pro-inflammatory cytokines where they combine with oxygen and protons to

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Metabolic disturbances in non-alcoholic fatty liver disease 555

form water. However, upstream of the production of dria [209]. Mitochondria and mtDNA are attractive
water, electrons react with oxygen to form free radicals vectors for the transmission of early environmental stress,
(superoxide anions). These radicals are dismutated by as they can harbour environmental damage for many cell
SOD (superoxide dismutase) to H2 O2 and then to divisions before cell dysfunction becomes evident [210].
water by glutathione peroxidase. Glutathione peroxidase Mitochondria depend on both nuclear and mtDNA to
requires GSH to function, and decreased GSH may function. mtDNA is sensitive to environmental changes
be accompanied by mitochondrial dysfunction and/or during oocyte maturation and during perimplantation
cell death [199]. In pathological conditions, it is development [211]. Therefore mitochondrial dysfunction
plausible that increased free radical production results in caused by changes in mtDNA or altered expression of
further mitochondrial damage and dysfunction, with a nuclear genes could cause both immediate and delayed
consequent decrease in oxidative capacity. A decrease in increases in oxidant stress which may alter nuclear gene
mitochondrial oxidative capacity could potentially result expression and cell function.
in an imbalance between fat oxidation and lipogenesis, As mentioned above, we have developed a mouse
leading to hepatic fat accumulation and NAFLD. model showing that manipulating the maternal dietary
exposure during pregnancy worsens development of the
adult offspring NAFLD phenotype. We have obtained
‘DEVELOPMENTAL ORIGINS’: ALTERED results from this model of the developmental origins of
EARLY DEVELOPMENT IN THE NAFLD of decreased mitochondrial complex activity.
PATHOGENESIS OF NAFLD To investigate the effect on the offspring of manipulating
the maternal diet during pregnancy, we have determined
Our group has published extensively in recent years whether there were changes in electron transport chain
showing that modification of intra-uterine nutrition enzyme complex activity in the liver from mouse
during gestation affects epigenetic regulation of key meta- offspring in adulthood which had been exposed to a
bolic genes and has the potential to modify the disease high-fat or to a control diet both pre- and post-natally
phenotype [200–208]. Specifically, our group has shown (K. D. Bruce, F. Cagampang and C. D. Byrne, unpub-
for the first time that prenatal nutrition induces differen- lished work). Female C57 BL/6J black mice were
tial changes to the methylation of individual CpG dinuc- randomly assigned to either a high-fat diet (HF, 45 %
leotides in the hepatic PPAR-γ promoter, altering mRNA kJ from fat, 20 % kJ from protein and 35 % kJ from
levels of the PPAR-α gene, downstream target genes and carbohydrate; n = 10) or to a standard laboratory chow
phenotype [202]. We have recently developed a mouse diet (C, 21 % kJ from fat, 18 % kJ from protein and 63 %
model of human NAFLD which shows that exposure to kJ from carbohydrate; n = 10). Dams were fed the diets
a high-fat diet in utero and during lactation exacerbates for 4 weeks prior to conception, during gestation and
the NAFLD phenotype exhibited in offspring that were lactation. At weaning, the offspring were assigned either
also fed a high-fat diet post-weaning (K. D. Bruce, the high-fat or control diets, generating four experimental
F. Cagampang and C. D. Byrne, unpublished work). groups: HF/HF (n = 12), HF/C (n = 12), C/HF (n = 12)
Thus with our findings showing an influence of intra- and C/C (n = 12), which represents the pre-natal/post-
uterine nutritional exposure to affect the risk of the natal diet respectively. In both male and female offspring,
NAFLD phenotype we suggest that intra-uterine nutri- body weights, total fat mass and systolic BP measure-
tion may have the potential to modify hepatic lipogenesis ments were significantly higher (P < 0.01) in offspring
through an influence of PPAR-γ and modifying SREBP- from HF/HF compared with the C/C or the C/HF
1c function. We speculate that increased dietary expo- groups. Hepatic electron transport chain complex activity
sure to a high-fat diet in the pregnant mother results in was decreased in offspring from HF/C and HF/HF
increased placental transfer of fatty acids to the fetus. groups compared with the C/C group, and the greatest
It is possible that increased placental transfer of fatty decrease was observed in Complex I activity, where a
acids to the fetus increases hepatic lipogenesis and 3.2- and 3.7-fold reduction in activity levels was seen
oxidative stress in the vulnerable fetal liver, perhaps also in HF/HF offspring (P < 0.05) and the HF/C offspring
increasing the demand for methyl donors to promote cell (P < 0.05) respectively, compared with C/C offspring.
growth and the subsequent development of NAFLD in These findings suggests that the pre-natal exposure to a
adulthood. It is conceivable that, if the increased demand high-fat diet impairs complex activity and mitochondrial
for methyl donors is not met, epigenetic changes in gene function. Steatosis was visible in liver sections (most
expression would result that predispose to conditions of marked in the HF/HF animals (Figure 7), HF/HF Oil red
fat accumulation and inflammation (such as NAFLD) in staining), and there was a marked inflammatory infiltrate
the developing offspring. in sections from the HF/HF group (Figure 7, HF/HF
The mitochondrial genome is particularly susceptible H&E staining) that is characteristic of human NASH.
to oxidative damage due to the absence of protective Thus these preliminary results provide evidence to
histones and incomplete repair mechanisms in mitochon- suggest that exposure to a high-fat diet in utero combined

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556 C. D. Byrne and others

Figure 7 Liver histology of a mouse model of the ‘developmental origins’ of NAFLD

Sections were stained with either (upper panels) haematoxylin and eosin (H&E) or (lower panels) Oil Red. Magnification, ×40. C, control diet; HF, high-fat diet.

with post-natal high-fat exposure contributes to a more This problem is now being addressed and there has
florid disease progression of fatty liver to NASH. been a marked improvement in an understanding of
Our group has also shown in human studies that the pathogenesis of NAFLD over the last decade. It
fatty liver is associated with increased NEFA supply to is now clear that factors operating outwith the liver
the liver [28,212,213], and we have shown in our mouse (e.g. uncontrolled adipocyte lipolysis) have a powerful
model that hepatic TAG is strongly correlated with CPT- impact to increase liver fat accumulation. There has been
1 expression (r = − 0.55, P = 0.033) [214], which mediates a marked improvement in our understanding of the role
the transfer of long-chain fatty acyl-CoAs across the of key transcription factors and the importance of three
mitochondrial membrane. CPT-1 is regulated by PPAR-α transcription factors SREBP-1c, LXR-α and ChREBP
activity and, thus, our finding that altered early nutrition operating in the liver to co-ordinate the regulation of
influences PPAR-α methylation [200,202] suggests that glycolysis and fatty acid synthesis. In the future, it
altered early nutrition may influence the flux of fatty may prove possible to modify the function of these
acids into the mitochondria for β-oxidation. We suggest transcription factors to ameliorate the development and
that increased flux of fatty acids to the developing fetal progression of NAFLD. At the present time, there is a
liver increases susceptibility of the liver to damage need to understand better the link between components
(NAFLD) induced by a second post-natal exposure to a of TAG synthesis and inflammation, as decreasing factors
high-fat diet. contributing to inflammation are likely to attenuate the
development of fibrosis and progression of disease. Work
from our laboratory suggests that factors affecting early
CONCLUSIONS fetal liver development, such as increased maternal dietary
fat consumption, may act to increase the vulnerability of
Precise estimates for the prevalence of NAFLD (and
developing fetal liver to fat accumulation in adulthood. If
the different subgroups such as NASH) are difficult to
the rapid progress in understanding of the pathogenesis of
obtain, because of an unmet need for simple diagnostic
NAFLD is maintained, it is likely that we will have novel
tests. Recent progress has been made in this field, and in
treatments for NAFLD and the tools for monitoring ther-
the near future it may be possible to use simple algorithms
apy without resorting to liver biopsy in the near future.
to replace liver biopsy for the diagnosis of NAFLD. It
has been shown recently that NASH is associated with
CHD, and in Type 2 diabetes NAFLD may be associated
with microvascular disease. To date there are no licensed ACKNOWLEDGEMENTS
treatments for NAFLD and the development of novel
therapies has been hampered by a poor understanding We thank Lucinda England for her help with the
of the molecular mechanisms contributing to NAFLD. manuscript.

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Metabolic disturbances in non-alcoholic fatty liver disease 557

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Received 27 June 2008/27 August 2008; accepted 7 October 2008

Published on the Internet 2 March 2009, doi:10.1042/CS20080253

C The Authors Journal compilation 
C 2009 Biochemical Society