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Drug Metab Rev. Author manuscript; available in PMC 2018 May 01.
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Published in final edited form as:

Drug Metab Rev. 2017 May ; 49(2): 197–211. doi:10.1080/03602532.2017.1293683.
Non-Alcoholic Fatty Liver Disease (NAFLD) - Pathogenesis,

Classification, and Effect on Drug Metabolizing Enzymes and
Transporters
Enoch Cobbina and Fatemeh Akhlaghi
Clinical Pharmacokinetics Research Laboratory, Department of Biomedical and Pharmaceutical
Sciences, University of Rhode Island, Kingston, RI, USA
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Abstract
Non-alcoholic fatty liver disease (NAFLD) is a spectrum of liver disorders. It is defined by the
presence of steatosis in more than 5 % of hepatocytes with little or no alcohol consumption.
Insulin resistance, the metabolic syndrome or type 2 diabetes and genetic variants of PNPLA3 or
TM6SF2 seem to play a role in the pathogenesis of NAFLD. The pathological progression of
NAFLD follows tentatively a ‘three-hit’ process namely steatosis, lipotoxicity and inflammation.
The presence of steatosis, oxidative stress and inflammatory mediators like TNF-α and IL-6 have
been implicated in the alterations of nuclear factors such as CAR, PXR, PPAR-α in NAFLD.
These factors may results in altered expression and activity of drug metabolizing enzymes (DMEs)
or transporters.
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Existing evidence suggests that the effect of NAFLD on CYP3A4, CYP2E1 and MRP3 are more
consistent across rodent and human studies. CYP3A4 activity is down-regulated in NASH whereas
the activity of CYP2E1 and the efflux transporter MRP3 are up-regulated. However, it is not clear
how the majority of CYPs, UGTs, SULTs and transporters are influenced by NAFLD either in
vivo or in vitro. The alterations associated with NAFLD could be a potential source of drug
variability in patients and could have serious implications for the safety and efficacy of
xenobiotics. In this review, we summarize the effects of NAFLD on the regulation, expression and
activity of major drug metabolizing enzymes and transporters. We also discuss the potential
mechanisms underlying these alterations.
Keywords
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Non-alcoholic fatty liver disease; steatosis; non-alcoholic steatohepatitis; diabetes; drug

metabolizing enzymes; transporters; cytochrome P450
Address for correspondence: Fatemeh Akhlaghi; Clinical Pharmacokinetics Research Laboratory; University of Rhode Island; Office
495 A; 7 Greenhouse Road; Kingston; RI 02881, USA. Phone: (401) 874 9205; Fax: (401) 874 5787; fatemeh@uri.edu.
Departmental Website URI: http://web.uri.edu/pharmacy/research/akhlaghi/
Laboratory Website URI: http://akhlaghilab.com/
Website Brown University: https://vivo.brown.edu/display/fakhlagh
Google Scholar: https://scholar.google.com/citations?user=wdnBYOAAAAAJ&hl=en
LinkedIn site: http://www.linkedin.com/in/akhlaghi
ORCID ID: orcid.org 0000-0002-3946-7615
Declaration of interest statement: Author EC and Author FA declare that they have no conflict of interest.
Cobbina and Akhlaghi Page 2
INTRODUCTION
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Non-alcoholic fatty liver disease (NAFLD) is a spectrum of liver disorders (Figure 1). It is a
condition defined by the presence of steatosis in more than 5 % of hepatocytes (Sanyal et al.,
2011) with little or no alcohol consumption. NAFLD consists of the benign non-alcoholic
fatty liver (NAFL), and the more severe non-alcoholic steatohepatitis (NASH). NASH is a
more progressive form of NAFLD and is characterized by steatosis, hepatocellular
ballooning, lobular inflammation and almost always fibrosis (Kleiner and Makhlouf, 2016).
In an effort to regenerate new cells, NASH progresses (Argo and Caldwell, 2009, Starley et
al., 2010) to cirrhosis with the hepatocytes replaced by scar tissues of type I collagen
produced by stellate cells. Cirrhosis is an end-stage organ failure that require liver
transplantation or may lead to hepatocellular carcinoma (Sorensen et al., 2003, Yasui et al.,
2011). With progression of NASH to full-blown cirrhosis, some of the histological
characteristics of NASH might be lost (Yoshioka et al., 2004).
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The metabolic syndrome, formerly known as Syndrome X, underlies both non-alcoholic

fatty liver disease (NAFLD) and diabetes. It is defined by the presence of at least three of the
following (Figure 2): abdominal obesity, increased triglycerides, reduced high density
lipoprotein (HDL) cholesterol, increased blood pressure and hyperglycemia (Alberti et al.,
2009). Insulin resistance appears to explain almost all situations of metabolic syndrome
(Eckel et al., 2010); and hence diabetes (Groop, 1999) and NAFLD (Marchesini et al.,
1999).
Though NAFLD is more prevalent in obese and diabetic patients, it is also present in lean
and non-diabetic individuals (Vos et al., 2011, Younossi et al., 2012). It is the most common
cause of cryptogenic cirrhosis (Clark and Diehl, 2003) and approximately 30–50 % of
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NASH patients may progress to cirrhosis within 10 years (Jou et al., 2008). NAFLD is not
only common in industrialized countries, but also developing ones. Global prevalence of
NAFLD has been reviewed and ranges from 6 – 35 % (Fazel et al., 2016, Sayiner et al.,
2016, Bellentani, 2017); and approximately 30% of the population of the United States (90
million persons) are estimated to be affected by NAFLD (Fazel et al., 2016). Eighteen out of
25 million Americans with diagnosed type 2 diabetes are believed to have NAFLD while
63–87% of patients having both diabetes and NAFLD may have NASH (Bazick et al., 2015,
Corey et al., 2016). The economic burden of NAFLD in four European countries (Germany,
France, Italy and the United Kingdom) was projected to be ~35 billion US dollars compared
to the approximately 103 billion dollars in the United States (Younossi et al., 2016).
Pharmacotherapy of NAFLD or NASH is an unmet clinical need. To date, no drug has

received FDA approval for NASH (Sanyal et al., 2015), thus a clinical or regulatory pathway
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has not yet been established. Current therapies like vitamin E (Rinella and Sanyal, 2016),
pentoxifylline (Zein et al., 2011) and insulin sensitizers such as pioglitazone in patients with
diabetes (Cusi, 2016) have been used. Therapies in development include obeticholic acid, a
semi-synthetic bile acid analogue undergoing development by Intercept Pharmaceuticals,
and elafibranor (formerly GFT505) a Peroxisome proliferator-activated receptor (PPAR)
alpha and a gamma agonist (Rinella and Sanyal, 2016). In view of the lack of standard
therapy, international guidelines on NAFLD (European Association for the Study of the
Liver (EASL), 2016) recommend lifestyle modifications particularly diet and exercise as
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viable treatment options. Recently, a role for Mediterranean diet in the prevention and
treatment of NAFLD has been proposed (Abenavoli et al., 2014, Godos et al., 2017).
The main clearance mechanisms of xenobiotics from the body are hepatic, renal and biliary.
It has been reported that more than 60 % of commonly prescribed drugs in the United States
are cleared hepatically (Williams et al., 2004), indicating the crucial role of the liver in drug
metabolism. Hepatic clearance of drugs is achieved through the activities of drug
metabolizing enzymes (DMEs) and transporters and hence factors that affect their regulation
and activities eventually alter drug disposition.
In this review, we summarize the effects of NAFLD on the regulation, expression and
activity of major drug metabolizing enzymes and transporters. In addition, we discuss the
various classification systems of NAFLD and the potential mechanisms underlying these
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alterations. Our review however does not include a discussion on models of NAFLD and
most findings published before 2011 since these have been reviewed by other groups
(Merrell and Cherrington, 2011, Naik et al., 2013).
Pathogenesis of NAFLD
The mechanisms leading to NAFLD is unclear to date. Several mechanisms have been
proposed, but insulin resistance seems to be pivotal in the pathogenesis of both NAFLD and
type 2 diabetes (Shulman, 2000, Tarantino and Finelli, 2013). The genetic variant of
PNPLA3 (patatin-like phospholipase domain containing 3), an enzyme encoding I148M
(rs738409 C/G) and involved in the hydrolysis of triacylglycerols in adipocytes, has been
reported to be associated with NAFLD independent of the metabolic syndrome (Romeo et
al., 2008, Sookoian and Pirola, 2011). Similarly, the genetic variant of the lipid transporter
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located on ER (endoplasmic reticulum) and ER-Golgi compartments, TM6SF2

(transmembrane 6 superfamily member 2), encoding E167K (rs58542926 C/T), causes loss
of function of the protein and increases hepatic deposition of triglycerides (Dongiovanni et
al., 2015). The pathological progression of NAFLD follows tentatively a ‘three-hit’ process
(Jou et al., 2008) namely steatosis, lipotoxicity and inflammation.
Steatosis results from the interplay between diet, gut microbiota (Jiang et al., 2015, Kirpich
et al., 2015), genetic factors (Romeo et al., 2008), and de novo lipogenesis via up-regulation
of lipogenic transcription factors like sterol regulatory binding protein-1c (SREBP1c),
carbohydrate-responsive element-binding protein (chREBP), and peroxisome proliferator-
activated receptor gamma (PPAR-γ) (Anderson and Borlak, 2008). Primarily, fatty acid (FA)
is stored in the adipose tissue as TAG (triacylglycerol). However, in obese subjects, fatty
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acids seem to be misrouted from their primary storage site to ectopic sites like skeletal and
hepatic tissues for re-esterification into diacyl glycerols (DAGs), perhaps through increased
adipocyte lipolysis. The uptake of fatty acid by these organs probably is facilitated by fatty
acid transport proteins (FATPs) and FAT/CD36 (fatty acid translocase) which have been
shown to be elevated in obese subjects and NAFLD patients (Greco et al., 2008, Fabbrini et
al., 2009).
Steatosis leads to increased signalling of the transcription factor NF-κβ (nuclear factor –
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kappaβ) through the upstream activation of IKKβ (inhibitor of nuclear factor kappaB [NF-
κB]). The activation of NF-κβ induces the production of pro-inflammatory mediators like
TNF-α (tumor necrosis factor - alpha), IL-6 (interleukin-6) and IL-1β (interleukin-1 β).
These cytokines contribute to the recruitment and activation of Kupffer cells (resident
hepatic macrophages) (Anderson and Borlak, 2008) to mediate inflammation in NASH
(Ramadori and Armbrust, 2001, Joshi-Barve et al., 2007). Additionally, TNF- α and IL-6
have been reported to play a role in hepatic insulin resistance through the up-regulation of
SOCS3 (suppressor of cytokine signalling 3) (Persico et al., 2007, Torisu et al., 2007).
The excess fat in the liver causes lipotoxicity and leads to organelle failure mainly
mitochondrial dysfunction and endoplasmic reticulum stress (Browning and Horton, 2004,
Bell et al., 2008 ). A dysfunctional mitochondrion has an elevated capacity to oxidize FA
resulting in the production of ROS (reactive oxygen species) and causing oxidative stress
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due to an imbalance between the production of ROS and protective oxidants. Oxidative
stress in NAFLD patients (Sanyal et al., 2001, Tiniakos et al., 2010) is regarded as the third
insult that eventually leads to hepatocyte death. The pathogenesis of NAFLD seem to be a
vicious cycle of steatosis, lipotoxicity and inflammation resulting in intricate alterations in
the histopathological and biochemical features of the liver.
Diagnosis and Classification of NAFLD

The diagnosis of NAFLD is challenging, as the current available routine techniques
(serological tests and imaging techniques) are unable to distinguish between steatosis and
NASH. Liver biopsy is considered the gold standard in defining NAFLD and is capable of
differentiating steatosis and NASH. It is however, not recommended for routine use due to
increased risk of bleeding and complications. In the last decades, many diagnostic non-
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invasive tools have been described (Table 1). Accurate diagnosis of NAFLD is important for
its classification. Some of the classification systems available include the scoring systems by
Matteoni (Matteoni et al., 1999 ), Brunt (Brunt et al., 1999), NASH CRN (Clinical Research
Network) system (Kleiner et al., 2005), and the SAF (steatosis, activity and fibrosis) system
(Bedossa et al., 2012). The different classification systems of NAFLD may thus yield
different results and hence introduce variability into scientific investigations.
One of the pioneering works with the largest number of patients and longest follow-up for
the stratification of NAFLD patients was carried out Matteoni and colleagues (Matteoni et
al., 1999 ). The Matteoni’s system was based on fat accumulation, inflammation, ballooning
degeneration, Mallory hyaline and fibrosis. NAFLD patients were put into four groups: Type
I (simple fatty liver), Type II (steatohepatitis), Type III (steatonecrosis) and Type IV
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(steatonecrosis plus either Mallory hyaline or fibrosis). Type I was relatively benign whereas
the necrotic forms were considered aggressive. The aggressive forms have higher risk of
cirrhosis and liver-related death. Though this system helps to identify patients at risk of
cirrhosis and liver-related death, it does not take into account NAFLD in children.
The system developed by Brunt (Brunt et al., 1999, Brunt et al., 2004) is semi-quantitative
and evaluates the unique lesions of NASH. It unifies steatosis and steatohepatitis into a
‘grade’ and fibrosis into a ‘stage’(Angulo, 2002). Steatosis is graded on a scale of 1 to 3
depending on the percentage of hepatocytes affected (<33 % =1; 33–66% = 2; >66% = 3).
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Steatohepatitis was similarly graded on a scale of 1 to 3 (1 = mild; 2 = moderate; 3 = severe)

but on the basis of the severity and extent of steatosis, ballooning, lobular inflammation and
portal inflammation. Fibrosis on the other hand was staged on a scale of 1 to 4. Brunt’s
system does not cover the entire spectrum of NAFLD as defined by Matteoni’s system.
Additionally, it was not designed to evaluate NAFLD in children (Kleiner et al., 2005).
In 2005, the Pathology Committee of the NASH Clinical Research Network (NASH CRN)
of the National Institute of Diabetes & Digestive & Kidney Disease (NIDDK) came up with
a scoring system and NAFLD activity score (NAS) for use in clinical trial (Kleiner et al.,
2005). The scoring system was intended to address the full spectrum of lesions of NAFLD.
The histological features considered were grouped into five broad categories each with a
scoring scale. These features, which were independently associated with NASH, included
steatosis (0–3), lobular inflammation (0–3), hepatocellular injury (0–2), fibrosis (0–4) and
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miscellaneous features like Mallory’s hyaline and glycogenated nuclei. The NAS is the
unweighted sum of steatosis, lobular inflammation, and hepatocellular ballooning scores.
NAS of ≥ 5 was found to correlate with the diagnosis of NASH and biopsies with scores of
less than 3 were classified as “not NASH”. Notwithstanding, not all biopsies with NAS ≥ 5
meet the diagnostic criteria of definite NASH and should be used carefully in establishing
the presence or absence of NASH (Brunt et al., 2011). In a number of experimental work
involving humans and rodents, a NAS score of at least 4 was considered as NASH (Canet et
al., 2014, Ferslew et al., 2015).
Recently, the SAF (steatosis, activity and fibrosis) system has been proposed. The SAF
considers steatosis, lobular inflammation and ballooning in defining NAFL and NASH. The
activity is defined as the sum of the grades of lobular inflammation and ballooning and
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ranges from 0–4. The presence of NAFLD is defined by steatosis in the presence of any
degree of activity. This implies that the definition of either NAFL or NASH requires the
presence of steatosis (1–3) and varying degree of activity (NAFL: steatosis (1–3) + lobular
inflammation (0) + ballooning (0–2), or steatosis (1–3) + lobular inflammation (1–2) +
ballooning (0); and NASH: steatosis (1–3) + lobular inflammation (1) + ballooning (1–2) or
steatosis (1–3) + lobular inflammation (2) + ballooning (1–2)) (Bedossa et al., 2012, Kleiner
and Makhlouf, 2016).
Clinicobiological scores have also been used in relation to NAFLD for several reasons
including selection of patients needing biopsy and prediction of advanced forms of NASH.
These clinicobiologial scores make use of indices like body mass index (BMI), Age,
AST/ALT ratio, albumin, platelet count, diabetes, hyperglycemia, insulin resistance index,
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triglycerides, hypertension and others (Angulo et al., 1999, Dixon et al., 2001, Harrison et
al., 2003). For instance, ‘BAAT’ scoring (Ratziu et al., 2000 ) uses BMI, age, ALT, and
serum triglycerides. The BAAT score is calculated as the sum of categorical variables with a
scale of 0 to 4. A score of 0 or 1 on the BAAT scale would indicate absence of septal
fibrosis. ‘HAIR’ scoring (Dixon et al., 2001) on the other hand utilizes hypertension, ALT
and insulin resistance as an index with a scale of 0 to 3. A score of ≥2 is suggestive of
NASH.
Possible mechanisms of the alteration of DMEs and transporters in NAFLD and diabetes
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The influence of diseases on DMEs and transporters is complex due to the associated
physiological and pathological changes. For instance, inflammatory conditions have been
reported to cause the release of circulating pro-inflammatory cytokines like TNF-α, IL-1β,
and IL-6 which act as signalling molecules to mediate the down-regulation of drug
metabolizing enzymes partly through the suppression of transcription (Aitken et al., 2006,
Aitken and Morgan, 2007). The inflammation models, bacteria endotoxemia
(lipopolysaccharide (LPS)) and turpentine have been employed in rodents and hepatocytes to
gain some insight into the role of cytokines on the regulation of DMEs and transporters. It
seems that in majority of cases, inflammation and the associated cytokines down-regulate
the expression and activity of DMEs and some transporters as described in these reviews
(Aitken et al., 2006, Morgan, 2009).
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Oxidative stress in NAFLD and diabetes causes activation of Nrf2 (nuclear factor erythroid
2-related factor 2) in both experimental (Fisher et al., 2008) and clinical studies (Hardwick
et al., 2010). Nrf2 is a specific transcription factor that controls the antioxidant response. It
is released from keapl (Kelch-like ECH-associated protein 1) and translocates to the nucleus
where it binds to antioxidant response element (ARE) within promoters of target genes, and
induces expression of DMEs and transporters central to the maintenance of oxidative stress
inducing molecules (Jaiswal, 2004, Nakata et al., 2006, Zhang, 2006).
Fatty acids regulate gene expression by controlling the activity or expression of key nuclear
receptors. In vitro studies have identified many transcription factors as possible targets for
fatty acid regulation, including hepatic nuclear factors (HNF-4α and γ), PPARα, β, γ1, and
γ2, SREBP-1c, retinoid X receptor (RXRα), liver X receptor (LXRα), and others. Some
nuclear receptors, PPAR, HNF4 (hepatic nuclear factor), RXRα, and LXRα, bind directly to
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non-esterified fatty acids (NEFA), but others like SREBP-1c and NF-κB are regulated by
fatty acids through indirect mechanisms (Jump et al., 2005, Jump, 2008). In rodents,
SREBP-1c inhibits PXR (pregnane X receptor) and CAR (constitutive androstane receptor)
(Roth et al., 2008), and has been shown to be up-regulated in obese insulin-resistant patients
(Pettinelli et al., 2009). The modulation of the activity of CAR and PXR by polyunsaturated
fatty acids (PUFA) has also been reported (Finn et al., 2009).
In addition, changes in the architecture of the liver in hepatic cirrhosis have been reported to
cause reduced liver blood flow, reduced functional hepatocytes and diminished functional
capacity of the liver to synthesize serum proteins including albumin (Elbekai et al., 2004,
Edginton and Willmann, 2008, Johnson et al., 2010). Collectively, the changes mediated by
excess fatty acids, cytokines, oxidative stress, and other mechanisms in NAFLD and diabetes
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may affect the hepatic metabolism of certain drugs possibly through the alteration of the
expression and activity of DMEs and transporters. This could result from host defence
mechanisms at the transcriptional as well as pre- and post-translational levels (George et al.,
1995, Renton, 2004, Aitken et al., 2006). These aberrant signals disrupt the normal hepatic
signalling pathways and eventually dysregulate major drug-metabolism-associated nuclear
factors leading to altered drug metabolism in NAFLD and diabetic patients (Naik et al.,
2013).
Hepatic Drug Metabolism

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Phase I reactions are mainly oxidative processes and are predominantly carried out by the
cytochrome P450 (CYP) enzyme system (Guengerich and MacDonald, 1990, Guengerich,
2008, Guengerich and Munro, 2013). Of the 18 known families of CYP enzymes (Zanger
and Schwab, 2013), only a few of the members belonging to families 1, 2 and 3 appear to be
relevant to biotransformation of xenobiotics (Cholerton et al., 1992, Zanger and Schwab,
2013). These include CYP1A1, CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9,
CYP2C19, CYP2D6, CYP2E1, CYP2J2, CYP3A4, and CYP3A5. Non-CYP enzymes
involved in phase I reactions include monoamine oxidase, flavin-containing monooxygenase
(Rettie et al., 1995, Fisher et al., 2002) and aldehyde oxidase (Johns, 1967 ).
Phase II biotransformation on the other hand are primarily conjugation reactions and it
includes glucuronidation (Meech and Mackenzie, 1997), sulfation (Negishi et al., 2001), and
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glutathione conjugation (Sofia et al., 1997). The enzymes responsible for these processes are
Uridine diphosphate (UDP) - glucuronosyl transferases (UGTs), Sulfotransferases (SULT),
and Glutathione -S-transferases (GSTs) respectively.
Drug transporters are crucial for metabolism of drugs and has been reviewed by several
groups (Giacomini et al., 2010). Hepatic transporters are classified into uptake and efflux
transporters (Mizuno and Sugiyama, 2002, Mizuno et al., 2003). The main uptake
transporters belong to the solute carrier (SLC) superfamily and facilitate the movement of
drugs into cells. These include OATPs (organic anion transporting polypeptides), OCTs
(organic cation transporter), and OATs (organic anion transporter). The efflux transporters
on the other hand belong to the ABC (ATP-binding cassette) superfamily and help move
drugs out of cells (Mizuno et al., 2003, Sugiura et al., 2006). Examples include P-gp (P-
glycoprotein), BCRP (Breast cancer resistance protein) and MRPs (Multidrug resistance-
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associated protein).
Several factors have been reported to affect DMEs and transporters. These include genetic
polymorphisms, epigenetic factors, and non-genetic factors. Genetic polymorphisms result
in alterations in DNA sequence of genes that regulate the expression of DMEs and
transporters; and have led to loss-of-function or gain-of-function variants. The association
between genetic polymorphisms and variation of plasma concentration levels of drugs as
well as response has been extensively studied (Koren et al., 2006, Elens et al., 2011).
Epigenetic influences on drug metabolism have also been reported. These are heritable
changes in gene function that are not based on DNA sequence variation, but covalent
modification of DNA, modification of histones or microRNA regulation (Pan et al., 2009,
Mohri et al., 2010). In addition to the above, non-genetic factors like sex (Schmidt et al.,
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2001, Wolbold et al., 2003), age (Cotreau et al., 2005, Stevens et al., 2008) and disease state
like diabetes (Dostalek et al., 2011, Dostalek et al., 2012a, Dostalek et al., 2012b) affect the
expression and activity of DMEs and transporters.
Effect of NAFLD on Phase I Drug Metabolizing Enzymes (DMEs)

CYP3A—This gene is part of a cluster of cytochrome P450 genes on chromosome 7q21.1
and includes four genes - 3A4, 3A5, 3A7 and 3A43 (Zanger and Schwab, 2013). It is the
most abundant human cytochrome P450 isoform in the liver and is involved in the
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metabolism of about half of clinically useful drugs (Guengerich, 1999). The CYP3A5
isoform is expressed mostly in Africans (Diczfalusy et al., 2011). It also exhibits wide inter-
individual variability in its expression and activity through polymorphisms, epigenetic and
non-genetic influences.
The influence of NAFLD on the expression and activity of CYP3A has been studied using
animal and cell culture models, human hepatic tissues, and human subjects (Woolsey et al.,
2015). Previous studies in rats and mice models are conflicting. However, a more consistent
result have been emerging showing down-regulation of the mRNA and protein expressions,
and the corresponding CYP3A activity in NAFLD (Table 2). This is perhaps due the use of
models that are able to simulate better the metabolic and histological lesions of NAFLD.
The activity of CYP3A decreased with severity of steatosis (Kolwankar et al., 2007) and
with the progression of NAFLD (Woolsey et al., 2015). Dostalek et al. (2011) observed
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significantly lower protein levels, reduced enzymatic activity of CYP3A4 and unchanged
mRNA levels in microsomal fractions of human diabetes mellitus livers (Dostalek et al.,
2011). Again, the plasma levels of atorvastatin, a substrate of CYP3A4 (Lennernäs, 2003),
has been reported to be elevated in patients with diabetes mellitus (Dostalek et al., 2012b).
In view of the high prevalence of NAFLD in the diabetic population, it is likely that NAFLD
could be involved in the down-regulation of CYP3A4 activity in the diabetic patients.
CYP3A genes seem to be regulated by a multiplicity of signalling pathways via CCAAT-

enhancer-binding proteins (C/EBP) (Martínez-Jiménez et al., 2005), HNF4 (Jover et al.,
2009), PXR (Liu et al., 2008), and CAR (Timsit and Negishi, 2007). A reduced CYP3A4
luciferase reporter activity in steatotic mice suggested a reduced CYP3A4 transcription in
NAFLD (Woolsey et al., 2015). The cytokine-mediated down-regulation of CYP3A4 (Werk
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and Cascorbi, 2014) in the course of the inflammatory response via the JAK/STAT (Janus
kinase/Signal Transducer and Activator of Transcription) pathway (Jover et al., 2002 ) seem
to be clinically relevant in NAFLD and diabetic patients due to circulating cytokines.
Additionally, it has been suggested that the hepatic CYP3A4 expression is probably down-
regulated by FGF21 (fibroblast growth factor 21) through the receptor-mitogen-activated
protein kinase (MAPK) pathway which leads to reduced gene transcription (Woolsey et al.,
2016).
CYP2—The CYP2 family contains several of the most important drug metabolizing CYPs
including CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, and CYP2D6. Some of these
members are highly polymorphic (Zanger and Schwab, 2013). The regulation of the
subfamilies of CYP2 appears to involve nuclear factors like PXR, CAR, GR, and HNF4α.
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Conflicting results have been reported in NAFLD and diabetic models. This is perhaps due
to differences in models used. Additionally, the polymorphic nature of some of the members
of this family could be a source of discrepancy in findings especially where the genotypes
involved are not considered. The effect of NAFLD on CYP2 enzymes has been studied by
several groups. Reduced activity and mRNA expression of CYP2A6, CYP2B6, CYP2C9
and CYP2D6 have been reported in primary human cultured hepatocytes exposed to
increasing concentrations (0.25 to 3 mM) of mixture (2:1) of oleic and palmitic acids
(Donato et al., 2006). This study suggested probable alterations in some of the CYP2
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enzymes in steatosis.
CYP2A6—CYP2A6 is clinically relevant for the hydroxylation of coumarin. The murine

ortholog of CYP2A6, Cyp2a5, was found to be elevated in the presence of steatosis (Li et
al., 2013, Cui et al., 2016) similar to the observations made in human hepatic tissues (Fisher
et al., 2009). These observations however contradict the observations made by another group
(Donato et al., 2006).
CYP2B6—CYP2B6 is an emerging enzyme with significant importance. It is involved in

the biotransformation of several clinically relevant drugs like bupropion, efavirenz and
cyclophosphamide. It also plays a role in the inactivation of environmental toxins. Recently,
in vivo and in vitro studies using male Sprague Dawley rats and rat hepatic tissues
respectively showed down-regulation of rat Cyp2b1(rat ortholog of human CYP2B6)
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activity, mRNA and protein expressions. This observation was made in both steatotic (HF
diet) and NASH (MCD-diet) models with pronounce effect in NASH. It appears progression
of NAFLD to hepatocellular carcinoma aggravates the decrease in CYP2B6 activity (Gao et
al., 2016). Notwithstanding, Fisher and colleagues (Fisher et al., 2009) observed a slight
increase in the mRNA levels, but did not observe any change in the protein level and activity
of CYP2B6 in steatotic and NASH human liver tissues. Since CYP2B6 is less abundant and
highly variable, evaluating the effect of heterogeneous NAFLD on its expression and activity
poses a challenge.
CYP2C—The CYP2C family of CYPs are responsible for the metabolism of about 12 %
(Wang and Tompkins, 2008) of clinically useful drugs. These include CYP2C8 (paclitaxel,
amodiaquine), CYP2C9 (warfarin, tolbutamide) and CYP2C19 (phenytoin, omeperazole).
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There seems to be very little information about the CYP2Cs since the last reviews on
NAFLD and DMEs (Merrell and Cherrington, 2011, Naik et al., 2013). The available reports
suggest alterations of CYP2C in NAFLD. However, the direction of change is not clear as
both increasing and decreasing trends have been observed (Fisher et al., 2009, Li et al.,
2016). The AUC of rosiglitazone, an insulin sensitizer and a substrate of CYP2C8 and
CYP2C9 (Baldwin et al., 1999), was found to be significantly increased in male mice after
high fat and high fructose NAFLD induction (Kulkarni et al., 2016). Nevertheless, it is not
clear whether this increase was mediated through down-regulation of the CYP2C8/9 or
alteration in transport mechanisms.
CYP2D6—CYP2D6 constitutes about 4 % of total CYP content, yet it is involved in the

biotransformation of more than 25 % (Wang and Tompkins, 2008) of clinically useful drugs
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including dextromethorphan and bufuralol. It is highly polymorphic (Ingelman-Sundberg,

2005) and the few reports are conflicting. In leptin-deficient (ob/ob) mice, the protein levels
of Cyp2d22 (rat ortholog of human CYP2D6) (Li et al., 2016) were decreased. Similarly, in
human liver tissues, CYP2D6 protein levels and activity showed a decreasing trend in NASH
(Fisher et al., 2009).
CYP2E1—CYP2E1 is the most studied CYP enzyme in relation to NAFLD. CYP2E1 is

involved in the biotransformation of acetaminophen, ethanol, acetone and fatty acid
oxidation. It is known for the generation of ROS like hydrogen peroxide, and superoxide
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anion radicals (Aubert et al., 2011) due to uncoupling of oxygen consumption with NADPH
(Nicotinamide adenine dinucleotide phosphate) oxidation and as a by-product of lipid
peroxidation (Robertson et al., 2001). It is therefore considered to probably worsen the
oxidative stress associated with diabetes and NAFLD, and may play a key role in the
progression of NAFLD (Aubert et al., 2011). In fact, it is suspected to be a contributor to
acetaminophen-induced liver injury in obesity and NAFLD (Michaut A1, 2014). There seem
to be an increasing number of findings in the literature to support the enhancement of
expression and activity of CYP2E1 in NAFLD in both humans and rodents (Chalasani et al.,
2003, Abdelmegeed et al., 2012, Aljomah et al., 2015). Results in rat studies have shown a
consistent trend of increase in Cyp2e1 expression and activity in MCD (Methionine choline
deficient) diet fed rats (Weltman et al., 1996). Diabetes has also been reported to increase the
mRNA and protein expressions of CYP2E1 (Lucas et al., 1998, Wang et al., 2003), and
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perhaps generating tissue-damaging hydroxyl radical in patients (Caro and Cederbaum,

2004).
CYP1A—The CYP1A subfamily has two functional members oriented head-to-head on

chromosome 15q24.1. These are CYP1A1 and CYP1A2 (Zanger and Schwab, 2013). The
two are highly inducible by ligands of CAR and AhR (aryl hydrocarbon receptor) (Zanger
and Schwab, 2013). CYP1A2 constitutes approximately 15 % of total hepatic CYP enzymes
(Wang and Tompkins, 2008). Its substrates include anticoagulants, antidepressants,
antihistamines and anticancer agents (Zanger and Schwab, 2013). Reports from different
groups about the down-regulation of CYP1A2 in NAFLD appears to be one of the most
consistent despite some discrepancies (Merrell and Cherrington, 2011). The levels of
expression of mRNA and protein are decreased in different rodent models of NAFLD
(Zhang et al., 2007, Hanagama et al., 2008). In human related tissues, down-regulation of
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mRNA, protein and activity have been observed (Donato et al., 2006, Fisher et al., 2009).
Significant increases in the systemic clearance of antipyrine and protein levels of hepatic
CYP1A2 were observed in diabetic rats possibly due to the enhancement of hepatic
CYP1A2-mediated metabolism (Ueyama et al., 2007). Similarly, the metabolism of
antipyrine was observed to be increased in patients with type 1 diabetes (Matzke et al.,
2000). The hepatic metabolism of theophylline into 1, 3- dimethyluric acid (3-DMU) by
CYP1A2 and CYP2E1 were studied using diabetes mellitus rat models (alloxan-induced and
streptozotocin-induced). A significant increase in the exposure of 1, 3-DMU was observed
in the diabetic rats compared to the controls. Based on in vitro rat hepatic microsomal
studies, the increased clearance of theophylline was confirmed in the diabetic rats (Kim et
al., 2005). Other studies in similar diabetic models have reported similar findings (Bae et al.,
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2006, DY et al., 2007).
Effect on Phase II Drug Metabolizing Enzymes (DMEs)

UDP-glucuronosyltransferases (UGTs)—Glucuronidation is the major route for phase
II reactions catalyzed by the UDP-glucuronosyltransferases (UGTs). UGTs have been
reported to be involved in the glucuronidation of more than 40 % of drugs in clinical use
(Wells et al., 2004). They are anchored in the endoplasmic reticulum. Members of the
UGT1A and 2B subfamilies appear relevant in humans due to their roles in the elimination
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of xenobiotics. In some reports, there was no change in Ugtb1 protein (rat) and UGT2B7
activity (humans) in NASH (Dzierlenga et al., 2015, Ferslew et al., 2015). An earlier work
utilizing human liver and kidney microsomes, however, observed a decrease in the activity
as well as reduction in the mRNA and protein expression of UGT2B7 in diabetes compared
to control (Dostalek et al., 2011 ). Again, it is not clear whether the presence of NASH in the
diabetic livers contributed to this observation. Limited literature on this subject matter does
not allow a clear understanding of how the expression and activities of UGTs are modified
by diabetes and NAFLD.
Sulfotransferases—Sulfotransferases (SULTs) are cytosolic enzymes that catalyze the

sulfonation reaction of xenobiotics and endogenous compounds by adding a sulfonate
moiety to a compound to increase its water solubility and decrease its biological activity. In
humans, three SULT families, SULT1, SULT2, and SULT4 have been reported. PPARα
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mediates the induction of human SULTs, thus implicating a role for fatty acids as
endogenous regulators of hepatic sulfonation in humans (Runge-Morris and Kocarek, 2005).
In human patients, SULT1A2 was found to be down-regulated in NASH (Younossi et al.,
2005); and resulted in decreased plasma levels of acetaminophen-sulfate (Canet et al., 2015).
Yalcin and colleagues (Yalcin et al., 2013) also observed that sulfotransferase activity
decreased significantly with severity of liver disease from steatosis to cirrhosis. Available
reports therefore suggest that the activities of SULT1A1 and SULT1A3 were lower in
disease states compared to non-steatotic tissues.
Glutatione-S-transferases—The Glutathione-S-transferases are present as different

isoforms - α (A=alpha), μ (M=mu), π (P=pi), θ (T=theta), and ζ (Z=zeta) (Hayes et al.,
2005). They are involved in the conjugation of glutathione (GSH) to reactive drug
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metabolites, though this reaction can be spontaneous without GST (Dragovic et al., 2010). A
number of studies into GST activity in NAFLD and diabetes have found decreased
enzymatic activity in ob/ob mice (Barnett et al., 1992, Roe et al., 1999) and human liver
samples (Hardwick et al., 2010). GSTM2, M4 and M5 expressions were higher in African
Americans with NASH than in Caucasians (Stepanova et al., 2010).
Effect of NAFLD on efflux and uptake transporters

The down-regulation of uptake and up-regulation of efflux transporters in obese and NAFLD
have been observed in studies involving rodents and human samples (Canet et al., 2014,
Canet et al., 2015). Though interspecies variation limits the use of rodents in modeling
human NAFLD, concordance analysis has suggested that both mouse and rat MCD models,
as well as mouse ob/ob and db/db NASH models show some similarity to human transporter
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mRNA and protein expression, and hence may be useful for predicting altered drug
disposition (Canet et al., 2014). Canet et al. (2014) observed mainly up-regulation of mRNA
and protein expressions of Mdr1 (multidrug resistance protein), Mrp1–4 (multidrug
resistance-associated protein) and Bcrp (Breast cancer resistance protein) in rat and mouse
NASH models. Conversely, the Oatps (organic anion transporting polypeptides) mainly
showed a down-regulation (Canet et al., 2014). The plasma concentrations of metformin, an
anti-hyperglycemic agent, were slightly increased in the WT/MCD and ob/Control groups.
In ob/MCD mice compared to Wild Type, the plasma concentrations were 4.8-fold higher.
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These changes were attributed to decreases in the kidney mRNA expression of Oct2 and
Mate1, the primary mediators of metformin elimination (Clarke et al., 2015).
In the literature, the influence of NAFLD on MRP2–3 appears more obvious compared to
other transporters (Hardwick et al., 2012, Canet et al., 2015). Table 3 shows some of the
published work on the effect of NAFLD on MRP3. In MCD diet-induced NASH male
Sprague-Dawley rats, mis-localization of Mrp2, the canaliculi efflux transporter, was
observed. Mrp2 appeared to pocket inward, resulting in a diminished function of effluxing
substrates into bile. On the other hand, the sinusoidal Mrp3 efflux transporter increased with
respect to protein expression leading to increased efflux of substrates into plasma
(Dzierlenga et al., 2015). These findings were consistent with human clinical studies
involving MRP3 and its morphine glucuronide (morphine 3 and 6 glucuronides) substrate in
NASH subjects (Ferslew et al., 2015). The AUC of morphine glucuronide was 58 % higher
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in NASH subjects compared to healthy subjects. The Cmax also was also significantly
higher in NASH subjects. In addition, fasting levels of total bile acids, glycocholate and
taurocholate were also elevated in NASH subjects suggesting up-regulation of the
basolateral efflux MRP-3 (Ferslew et al., 2015).
Clinical impact of NAFLD/NASH on pharmacotherapy

Though very few clinical studies have reported the impact of NAFLD on pharmacotherapy,
they strongly highlight the potential of NAFLD to cause variable drug response, adverse
drug reaction and eventually toxicity through alteration of pharmacokinetic profile.
Midazolam (Woolsey et al., 2015), morphine (Ferslew et al., 2015) and acetaminophen
(Canet et al., 2015) have been evaluated in both healthy and NAFLD patients. NAFLD seem
to increase the AUC of midazolam by reducing the activity of CYP3A4; and similarly
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increase the AUC of the glucuronide metabolites of morphine and acetaminophen via the up-
regulation of the MRP3 efflux transporter. Perhaps, the available evidence in the literature is
the main motivation behind the emerging interest in drug disposition in NAFLD patients.
Hopefully, more clinical studies would be conducted to gain more insight into the nature and
extent of impact of NAFLD on pharmacotherapy.
Challenges to studying the effect of NAFLD on DMEs and Transporters

Studying the effect of NAFLD is challenging. First, the pathogenesis of NAFLD is not
clearly understood, and is usually asymptotic requiring biopsy for definitive diagnosis. Due
to ethical reasons, researchers are unable to routinely obtain biopsies from patients for
studies. In addition, the presence of co-morbidities particularly diabetes, which is highly
prevalent in NAFLD patients, is not accounted for. For instance, it has been demonstrated
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that antipyrine elimination rate was dependent on the type of diabetes (type 1 versus type 2)
and gender (Sotaniemi et al., 2002). It was observed that insulinopenia enhanced hepatic
microsomal enzyme activity (probably through increased ketobodies), whereas relative
insulin deficiency was associated with decreased metabolic activity (Sotaniemi et al., 2002).
Since the presence of diabetes and other demographic characteristics could confound the
effect of NAFLD on DMEs and transporters, it may be necessary to account for them.
Finally, the absence of consensus on NASH models and NAFLD classification system to use
for experiments has permitted the use of different NASH models and classification systems.
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For instance, a mice diabetic model of NASH only recapitulated human CYP alterations in
NAFLD partially (Li et al., 2016); and hence may be inadequate for all CYPs. This has
made comparison of results from some groups difficult. It is anticipated that as research
advances in this area, these procedures would be harmonized to allow comparability of
results.
CONCLUSION
NAFLD and diabetes are gradually becoming pandemic globally. Limited options are
available for the treatment of NASH; hence, several pharmaceutical companies are trying to
develop new molecules for this condition. However, lack of knowledge on the effect of
NAFLD or NASH on the expression and activity of hepatic DMEs and transporters can
impede drug development in this area. Current research findings, though limited and
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sometimes conflicting, suggest alterations in DMEs and transporters in NAFLD. Few of the
results however are consistent across studies and species and includes the down-regulation
of CYP3A; and up-regulation of CYP2E1 and MRP3. Results from other DMEs and
transporters are either lacking or conflicting. Investigating the influence of NAFLD on
DMEs and transporters is challenging because NAFLD is heterogeneous and involves a
spectrum of hepatic lesions. The challenges introduce another layer of variability to NAFLD
experimental studies. The presence of steatosis, oxidative stress and inflammatory mediators
like TNF-α and IL-6 have been implicated in the alterations of nuclear factors in NAFLD.
Consequently, the regulation of transcription factors like CAR, PXR, PPAR-α, etc. may
change and eventually alter the expression of DMEs and transporters. These alterations
could be potential sources of drug variability in patients and could have serious
consequences on safety and efficacy. We recommend more studies in this area to augment
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our understanding on the effect of NAFLD on drug metabolism.
Acknowledgments
Support of grant # R15 GM101599 from the National Institutes of Health is gratefully acknowledged.
ABBREVIATIONS
3-DMU 1, 3- dimethyluric acid
ABC ATP-binding cassette
AhR Aryl hydrocarbon receptor

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ALT Alanine transaminase
ARE Antioxidant response element
AST Aspartate transaminase
AUC Area under the curve
BAAT BMI, Age, ALT, Triglycerides
BCRP Breast cancer resistance protein

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BMI Body Mass Index
C/EBPs CCAAT-enhancer-binding proteins (or C/EBPs)
CAR Constitutive androstane receptor
chREBP Carbohydrate-responsive element-binding protein
CT Computerized tomographic
CYP Cytochrome P450
DAGs Diacyl glycerols
DME Drug metabolizing enzyme

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ER Endoplasmic Reticulum
FAT/CD36 Fatty acid translocase
FATPs Fatty acid transport proteins
FGF21 Fibroblast growth factor 21
GR Glucocorticoid receptor
GSTs Glutathione -S-transferases
HAIR Hypertension, ALT, Insulin resistance

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HDL High-density lipoprotein
HFD High fat diet
HNF-4 Hepatic nuclear factors 4
IL-1β Interleukin-1 β
IL-6 Interleukin-6
JAK/STAT Janus kinase/Signal Transducer and Activator of Transcription
keapl Kelch-like ECH-associated protein 1
LPS Lipopolysaccharide
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LXRα Liver X receptor alpha
MAPK Mitogen-activated protein kinase
MCD Methionine choline deficient
MRI Magnetic resonance imaging
MRP Multidrug resistance-associated protein
NADPH Nicotinamide adenine dinucleotide phosphate (reduced)

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NAFL Non-alcoholic fatty liver
NAFLD Non-alcoholic fatty liver disease
NAS NAFLD activity score
NASH Non-alcoholic steatohepatitis
NASH CRN NASH Clinical Research Network
NEFA Nonesterified fatty acids
NF-κβ Nuclear factor – kappaβ
NIDDK National Institute of Diabetes & Digestive & Kidney Disease

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Nrf2 Nuclear factor erythroid 2–related factor 2
OATPs Organic anion transporting polypeptides
OATs Organic anion transporter
OCTs organic cation transporter
P-gp Permeability glycoprotein or P-glycoprotein
PNPLA3 Patatin-like phospholipase domain containing 3
PPARα Peroxisome proliferator-activated receptor alpha

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PPAR-γ Peroxisome proliferator-activated receptor gamma
PUFA Polyunsaturated fatty acids
PXR Pregnane X receptor
QUICKI Quantitative insulin sensitivity check index
RXRα Retinoid X receptor alpha
SAF Steatosis, activity and fibrosis
SLC Solute carrier superfamily
SOCS3 Suppressor of cytokine signalling 3

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SREBP1c Sterol regulatory binding protein-1c
SULT Sulfotransferases
TAG Triacylglycerol
TM6SF2 Transmembrane 6 superfamily member 2
TNF-α Tumor necrosis factor - alpha
UGTs Uridine diphosphate (UDP) - glucuronosyl transferases

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Figure 1.
The progressive stages of NAFLD (non-alcoholic fatty liver disease). The benign form of
NAFLD, NAFL (non-alcoholic fatty liver), progresses to NASH (non-alcoholic
steatohepatitis) with or without fibrosis. Subsequently, NASH leads to cirrhosis and
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eventually hepatocellular carcinoma (HCC). NASH may progress to HCC without cirrhosis.
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Figure 2.
Major components of the metabolic syndrome. The presence of at least three of these
components define the presence of metabolic syndrome.
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Table 1
Biomarkers and imaging techniques employed in diagnosis of NAFLD.

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Diagnosis Tools Technique/Principle Features References

Serological Tests Aspartate aminotransferase (AST) Raised levels not indicative of NAFLD (Mofrad et al., 2003,
because AST and ALTs are normal in Browning et al., 2004,
Alanine aminotransferase (ALT) some NAFLD patients. Bugianesi et al., 2004)
AST/ALT > 1 is predictive of fibrosis
Imaging Techniques Ultrasonography Sensitive when steatosis is > 30 % of (Wieckowska and Feldstein,
hepatocytes; Does not distinguish 2008)
between steatosis and NASH
Computerized Tomographic (CT) More sensitive than ultrasonography

Scanning Cannot distinguish between steatosis
Magnetic Resonance Imaging (MRI) and NASH
Expensive
Transient Elastography Can detect fibrosis but expensive
Liver Biopsy Histological evaluation of hepatic tissues. Gold Standard but invasive and may be (Ratziu et al., 2005,
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Hepatic lesions like steatosis, involved with complications and Wieckowska and Feldstein,
inflammation and ballooning are graded; sampling variability 2008)
and fibrosis is staged. Able to detect steatosis and
inflammation
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Table 2
The effect of NAFLD on CYP3A4/CYP3A5. Overall, NAFLD progression seem to reduce the activity of CYP3A.
Study NAFLD Model NAFLD category mRNA Protein Activity Activity Probe
(Kolwankar et al., 2007) Human liver tissues (Ex vivo) Steatosis Decreased Slight decrease Decreased Testosterone
(Fisher et al., 2009) Human liver tissues (Ex vivo) Steatosis No change Slight increase Decreased Testosterone
Cobbina and Akhlaghi
NASH (fatty) No change Decreased Decreased
NASH (not fatty) No change Decreased Decreased
(Woolsey et al., 2015) Human Subjects (in vivo) Steatosis Not Reported Not reported Decreased (2.4 fold) Midazolam
NASH Decreased (2.5 fold)
Human liver tissues (Ex vivo) Steatosis Decreased (60 %) Not reported Not reported
NASH Decreased (69 %)
Female Mice (in vivo) HFD Steatosis Not reported Not reported Not reported CYP3A4 Luciferase Reporter plasmid
Huh7 hepatoma cells (in vitro) Steatosis Decreased (80 %) Not reported Decreased (38 %) Midazolam
(Li et al., 2016) ob/ob male Mouse (in vivo) (MCD) Diabetic Increase Slight decrease Slight decrease Midazolam
Diabetic NASH Increase Decreased Slight decrease
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Table 3
The effect of NAFLD on MRP3. Overall, NAFLD progression seem to increase the expression and activity of MRP3
Study Species Ref/NAFLD Endpoint Change Probe Substrate

(Hardwick et al., 2012) Rats (male Sprague-Dawley) Control/NASH mRNA level Significantly increased
Protein Significantly increased

Cobbina and Akhlaghi
Plasma Concentration Significantly increased Ezetimibe glucuronide
(Dzierlenga et al., 2015) Rats (male Sprague-Dawley) Control/NASH AUC 150 % increase Morphine glucuronide
Protein Significantly increased
Activity Significantly increased
(Ferslew et al., 2015) Human Healthy/NASH Cmax 52 % increase in NASH Morphine glucuronide
AUC 58 % increase in NASH Morphine glucuronide
(Canet et al., 2015) Human (Children) Healthy/Steatosis/NASH AUC Increased Acetaminophen glucuronide
Human Liver Tissues Healthy/Steatosis/NASH MRP3 Protein Significantly increased
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Non-Alcoholic Fatty Liver Disease (NAFLD) - Pathogenesis,

Non-alcoholic fatty liver disease; steatosis; non-alcoholic steatohepatitis; diabetes; drug

The metabolic syndrome, formerly known as Syndrome X, underlies both non-alcoholic

Pharmacotherapy of NAFLD or NASH is an unmet clinical need. To date, no drug has

located on ER (endoplasmic reticulum) and ER-Golgi compartments, TM6SF2

Diagnosis and Classification of NAFLD

Steatohepatitis was similarly graded on a scale of 1 to 3 (1 = mild; 2 = moderate; 3 = severe)

Hepatic Drug Metabolism

Effect of NAFLD on Phase I Drug Metabolizing Enzymes (DMEs)

CYP3A genes seem to be regulated by a multiplicity of signalling pathways via CCAAT-

CYP2A6—CYP2A6 is clinically relevant for the hydroxylation of coumarin. The murine

CYP2B6—CYP2B6 is an emerging enzyme with significant importance. It is involved in

CYP2D6—CYP2D6 constitutes about 4 % of total CYP content, yet it is involved in the

including dextromethorphan and bufuralol. It is highly polymorphic (Ingelman-Sundberg,

CYP2E1—CYP2E1 is the most studied CYP enzyme in relation to NAFLD. CYP2E1 is

perhaps generating tissue-damaging hydroxyl radical in patients (Caro and Cederbaum,

CYP1A—The CYP1A subfamily has two functional members oriented head-to-head on

2006, DY et al., 2007).

Effect on Phase II Drug Metabolizing Enzymes (DMEs)

Sulfotransferases—Sulfotransferases (SULTs) are cytosolic enzymes that catalyze the

Glutatione-S-transferases—The Glutathione-S-transferases are present as different

Effect of NAFLD on efflux and uptake transporters

Clinical impact of NAFLD/NASH on pharmacotherapy

Challenges to studying the effect of NAFLD on DMEs and Transporters

our understanding on the effect of NAFLD on drug metabolism.

ABC ATP-binding cassette

AhR Aryl hydrocarbon receptor

ALT Alanine transaminase

ARE Antioxidant response element

AST Aspartate transaminase

AUC Area under the curve

BAAT BMI, Age, ALT, Triglycerides

BCRP Breast cancer resistance protein

BMI Body Mass Index

C/EBPs CCAAT-enhancer-binding proteins (or C/EBPs)

CAR Constitutive androstane receptor

chREBP Carbohydrate-responsive element-binding protein

CYP Cytochrome P450

DAGs Diacyl glycerols

DME Drug metabolizing enzyme

FAT/CD36 Fatty acid translocase

FATPs Fatty acid transport proteins

FGF21 Fibroblast growth factor 21

GSTs Glutathione -S-transferases

HAIR Hypertension, ALT, Insulin resistance

HDL High-density lipoprotein

HFD High fat diet

HNF-4 Hepatic nuclear factors 4

JAK/STAT Janus kinase/Signal Transducer and Activator of Transcription

keapl Kelch-like ECH-associated protein 1

LXRα Liver X receptor alpha

MAPK Mitogen-activated protein kinase

MCD Methionine choline deficient

MRI Magnetic resonance imaging

MRP Multidrug resistance-associated protein

NADPH Nicotinamide adenine dinucleotide phosphate (reduced)

NAFL Non-alcoholic fatty liver

NAFLD Non-alcoholic fatty liver disease