International Journal of

Molecular Sciences

Review
The Role of Insulin Resistance and Diabetes in
Nonalcoholic Fatty Liver Disease
Hideki Fujii 1 , Norifumi Kawada 2, * and Japan Study Group of NAFLD (JSG-NAFLD) †
1 Department of Premier Preventive Medicine, Graduate School of Medicine, Osaka City University,
Osaka 545-8585, Japan; rolahideki@med.osaka-cu.ac.jp
2 Department of Hepatology, Graduate School of Medicine, Osaka City University, Osaka 545-8585, Japan
* Correspondence: kawadanori@med.osaka-cu.ac.jp; Tel.: +81-6-6645-3897
† Members are listed in the Acknowledgments.




Received: 18 April 2020; Accepted: 24 May 2020; Published: 29 May 2020


Abstract: Nonalcoholic fatty liver disease (NAFLD) consists of the entire spectrum of fatty liver disease
in patients without significant alcohol consumption, ranging from nonalcoholic fatty liver (NAFL)
to nonalcoholic steatohepatitis (NASH) to cirrhosis, with NASH recently shown as an important
cause of hepatocellular carcinoma (HCC). There is a close relationship between insulin resistance
(IR) and NAFLD, with a five-fold higher prevalence of NAFLD in patients with type 2 diabetes
(T2DM) compared to that in patients without T2DM. IR is involved in the progression of disease
conditions such as steatosis and NASH, as well as hepatic fibrosis progression. The mechanisms
underlying these processes involve genetic factors, hepatic fat accumulation, alterations in energy
metabolism, and inflammatory signals derived from various cell types including immune cells.
In NASH-associated fibrosis, the principal cell type responsible for extracellular matrix production
is the hepatic stellate cell (HSC). HSC activation by IR involves “direct” and “indirect” pathways.
This review will describe the molecular mechanisms of inflammation and hepatic fibrosis in IR,
the relationship between T2DM and hepatic fibrosis, and the relationship between T2DM and HCC in
patients with NAFLD.

Keywords: hepatic fibrosis; insulin resistance; inflammation; stellate cell; hepatocellular carcinoma

1. Introduction
Nonalcoholic fatty liver disease (NAFLD) is a major form of chronic liver disease that affects both
adults and children worldwide [1]. It is one of the clinical consequences of obesity and can progress to
nonalcoholic steatohepatitis (NASH), which is pathologically characterized by the presence of steatosis,
inflammation, and fibrosis in the liver parenchyma, which ultimately leads to cirrhosis, hepatocellular
carcinoma (HCC), and end-stage liver failure [1–3]. The prevalence of NAFLD in the world population
is estimated to be 25%, whereas the pooled overall NASH prevalence estimated among biopsied NAFLD
patients is 59% [4,5]. Recent meta-analysis revealed that over 2146 person-years of follow-up evaluation,
34% had fibrosis progression, 43% had stable fibrosis, and 22% had an improvement in fibrosis stage [6].
The listed diagnosis in the United Network for Organ Sharing database suggests that since 2000,
the number of patients with NASH cirrhosis has increased, whereas that of patients with cryptogenic
cirrhosis has decreased [7]. Liver-specific mortality and overall mortality among NAFLD and NASH
patients were 0.77 and 11.77 per 1000 person-years, and 15.44 and 25.56 per 1000 person-years,
respectively [4]. Patients with NAFLD cirrhosis (F4) predominantly develop liver-related events,
whereas those with bridging fibrosis (F3) predominantly develop non-hepatic cancers and vascular
events [8]. Estimates of NAFLD heritability range from 20% to 70%, with an estimated shared genetic

Int. J. Mol. Sci. 2020, 21, 3863; doi:10.3390/ijms21113863 www.mdpi.com/journal/ijms

Int. J. Mol. Sci. 2020, 21, 3863 2 of 17

effect or determination between steatosis and fibrosis of 75% [9], depending on ethnicity, study design,
environmental factors, and methodology used for NAFLD characterization [10,11].
Recent evidence showed that extra-hepatic organs influence NAFLD progression [12–15].
Progressive adipose tissue dysfunction and insulin resistance (IR) are key events in NASH development,
supporting the existence of an adipose tissue–liver crosstalk [12,13]. Most recently, Vily-Petit et al.
reported that increased intestinal gluconeogenesis (IGN) improves glucose control and prevents the
onset of hyperglycemia under a high-fat/high-sucrose diet [14]. Conversely, the suppression of IGN
promotes lipid accumulation in the liver even under a standard diet. IGN provides metabolic benefits
by initiating a gut–brain neural signal, thus triggering brain-dependent regulations of peripheral
metabolism [15]. The authors suggested that IGN can specifically modulate the onset of hepatic
steatosis and molecular events associated with the development of NAFLD through a gut–brain–liver
neural circuit [14].
NAFLD is driven by ectopic fat accumulation in the liver, is an indicator of IR, and signals the
possibility of ectopic fat accumulation in inappropriate parts of the body, such as in intramuscular,
perivascular, and pericardial regions, and visceral fat deposition [16]. Therefore, NAFLD is often
described as the hepatic manifestation of metabolic syndrome and is a major risk factor for type 2 diabetes
mellitus (T2DM), in which NAFLD is commonly found as a comorbidity [16,17]. Recent meta-analysis
showed that the overall prevalence of NAFLD among patients with T2DM is 55.5%, whereas the global
prevalence of NASH among patients with T2DM is 37.3% [18]. Compared to western populations,
Asians are particularly susceptible to NAFLD partially because of body composition differences in
fat and muscle, as well as genetic susceptibility through the predisposition to T2DM, patatin-like
phospholipase domain containing 3 (PNPLA3) SNPs, and polymorphisms in apolipoprotein 3 [3].
T2DM is an important risk factor for NAFLD and seems to accelerate the progression of liver disease in
NAFLD patients [1–4,8,16–18]. In this review, we discuss the most updated data characterizing the
role of IR and T2DM in the progression of hepatic fibrosis in NAFLD. As described later, improving IR
can be one of the potential therapeutic approaches for NASH fibrosis, even in the absence of T2DM.
This is an innovative review because we discussed the influence of IR on the progression of hepatic
fibrosis in NAFLD, both in basic and clinical aspects, for the first time. Some of the studies reported in
this review analyzed data from the Japan Study Group of NAFLD (JSG-NAFLD).

2. Molecular Mechanism of Inflammation in IR

Cell death and inflammation are key drivers of fibrosis in NASH and other forms of chronic
liver disease [19,20]. The primary pathophysiological mechanisms of IR induced by inflammatory
mediators are probably the result of interference with insulin signaling [17]. Insulin acts in
all cells by binding to its specific receptor and activating a cascade of intracellular signaling.
Upon insulin binding, the insulin receptor phosphorylates itself and several members of the
insulin receptor substrate (IRS) family. IRS1 and IRS2 are the main mediators of insulin signaling
in the liver, where they control insulin sensitivity [17,21,22]. The canonical IRS signaling
pathways include the IRS1- or IRS2-dependent signaling pathways that use the activities of
phosphatidylinositol 3-kinase (PI3K)-phosphoinositide-dependent kinase (PDK)-protein kinase B
(AKT) and the RAS−extracellular-signal regulated kinase (ERK) [17,21]. The PI3K-PDK-AKT pathway
mediates gluconeogenesis and glycogen synthesis. Additionally, the RAS-ERK pathway mediates cell
proliferation and survival [21]. IR is defined as the impairment of the appropriate downstream effects
of insulin signaling in target tissues, primarily the liver, muscle, and adipose tissue. IR is postulated to
begin in muscle tissue, which accounts for up to 70% of glucose disposal, whereas immune-mediated
inflammatory change and excess free fatty acids (FFA) cause ectopic lipid deposition [23].
IR in adipose tissue results in increased lipolysis in adipocytes and an increased circulating FFA,
which further exacerbates steatosis and IR in muscle tissue. During caloric intake, insulin reduces
hepatic glucose production by inhibiting glycogenolysis and limiting the postprandial rise in glucose.
In IR, this feedback mechanism is impaired and hepatic glucose production continues to rise even
Int. J. Mol. Sci. 2020, 21, 3863 3 of 17

when postprandial glucose increases. Glucotoxicity is associated with elevated glucose levels and
further contributes to IR [23].
Int. J. Mol. Sci. 2019, 20, x FOR PEER REVIEW 3 of 18
Pro-inflammatory cytokines and transcription factors are highly expressed in various tissues,
includingPro-inflammatory
adipose tissue or cytokines
liver inand transcription
obesity factors
and related are highlySeveral
disorders. expressed in various tissues,
inflammatory cytokines,
including
such as adipose tissue
tumor necrosis factor or liver inor
(TNF)-α obesity and related
interleukin (IL)-6,disorders. Several
can activate inflammatory
the inhibitor cytokines,
of nuclear factor-κB
such as tumor necrosis factor (TNF)-α or interleukin (IL)-6, can activate
kinase (IKK) complex IKKβ, c-Jun N-terminal kinase (JNK), which is also known as mitogen-activated the inhibitor of nuclear
factor-κB kinase (IKK) complex IKKβ, c-Jun N-terminal kinase (JNK), which is also known as
protein kinase. Additionally, suppressor of cytokine signaling (SOCS) can phosphorylate IRS1 and
mitogen-activated protein kinase. Additionally, suppressor of cytokine signaling (SOCS) can
IRS2 to inhibit insulin signaling [17,22]. Hyperglycemia can increase oxidative stress [22], which can
phosphorylate IRS1 and IRS2 to inhibit insulin signaling [17,22]. Hyperglycemia can increase
inhibit insulin signaling through the activation of inhibitor of nuclear factor kappa-B (I-κB) kinase
oxidative stress [22], which can inhibit insulin signaling through the activation of inhibitor of nuclear
subunit
factorbeta (IKKβ)
kappa-B andkinase
(I-κB) JNK. IKKβ
subunitand betaJNK canand
(IKKβ) activate nuclear
JNK. IKKβ andfactor
JNK cankappa-B
activate(NF-κB) and cause
nuclear factor
its translocation
kappa-B (NF-κB) to theandnucleus.
cause its [17]. Endoplasmic
translocation reticulum
to the nucleus. [17].(ER) stress can
Endoplasmic also activate
reticulum the JNK
(ER) stress
pathway
can also[17,24]. Thethe
activate inflammatory
JNK pathway pathways
[17,24]. Thethat affect IR are
inflammatory summarized
pathways inIR
that affect Figure 1.
are summarized
in Figure 1.

Figure 1. Summary of inflammatory pathways affecting hepatic insulin resistance (IR) in nonalcoholic
Figure 1. Summary of inflammatory pathways affecting hepatic insulin resistance (IR) in nonalcoholic
fatty fatty
liverliver
disease (NAFLD).
disease (NAFLD). Insulin
Insulinactivates
activates its
its receptor, whichresults
receptor, which results
in in tyrosine
tyrosine phosphorylation
phosphorylation
on theoninsulin receptor
the insulin receptorsubstrate
substrate(IRS1
(IRS1and
and IRS2) and activation
IRS2) and activationofof downstream
downstream effector
effector pathways,
pathways,
including the phosphatidylinositol
including the phosphatidylinositol 3-kinase (PI3K)-phosphoinositide-dependent
3-kinase (PI3K)-phosphoinositide-dependent kinase
kinase(PDK)-protein
(PDK)-
kinase B (AKT)
protein andB the
kinase (AKT)RAS−extracellular-signal-regulated kinase (ERK)
and the RAS−extracellular-signal-regulated kinase pathways (i.e., canonical
(ERK) pathways (i.e.,
canonical IRS
IRS signaling). signaling).
Numerous Numerous pro-inflammatory
pro-inflammatory signaling orsignaling or reactive
reactive oxygen oxygen
species canspecies
activatecan IKK-β.
activate IKK-β.
The activated NF-kBThe activated
is then NF-kB is into
translocated then the
translocated into binds
nucleus and the nucleus and binds
to specific DNAtoresponse
specific DNA
elements.
response elements.
Inflammatory cytokinesInflammatory cytokines IR
such as IL-6 promote such as IL-6 promote
by inducing IR byofinducing
suppressor cytokinesuppressor of
signaling (SOCS)
cytokine signaling (SOCS) 1 and 3. SOCS1 and SOCS3 impair insulin signaling through ubiquitin-
1 and 3. SOCS1 and SOCS3 impair insulin signaling through ubiquitin-dependent degradation of IRS.
dependent degradation of IRS. The c-Jun N-terminal kinase (JNK, or mitogen-activated protein
The c-Jun N-terminal kinase (JNK, or mitogen-activated protein kinase) represents another important
kinase) represents another important inhibitory kinase of IRS that is activated in response to a variety
inhibitory kinase of IRS that is activated in response to a variety of extracellular stimuli and cellular
of extracellular stimuli and cellular stressors such as oxidative and endoplasmic reticulum (ER) stress.
stressors such as oxidative and endoplasmic reticulum (ER) stress.
From a clinical point of view, data on the changes in hepatic expression of IRS1 and IRS2 with
From a clinical point
the pathological of of
severity view, datainonpatients
NAFLD the changes in hepatic
are insufficient expression
and of IRS1
do not provide and IRS2 with
a consensus.
the pathological
Rametta et al.severity
reportedofthat
NAFLD
hepaticinIRS1
patients
mRNA arelevels
insufficient
did not and dobut
differ, notIRS2
provide
mRNA a consensus.
levels
Rametta et al. reported
progressively increasedthat hepatic
with IRS1 ofmRNA
the severity levels[25].
the histology didConversely, but IRS2
not differ,Honma et al. mRNA
examinedlevels
progressively increased
mRNA expression in with the severity
51 human of the
liver biopsy histology
samples [25].from
obtained Conversely,
nondiabeticHonma et al.
subjects [26].examined
The
hepatic expression of IRS1 was unchanged in NAFLD conditions, whereas
mRNA expression in 51 human liver biopsy samples obtained from nondiabetic subjects the expression of IRS2 in [26].
NASH samples was lower than that in health control samples. Moreover,
The hepatic expression of IRS1 was unchanged in NAFLD conditions, whereas the expression the severity of lobular
inflammation
of IRS2 was negatively
in NASH samples correlated
was lower than with hepatic
that in healthIRS2 mRNA
control levels (rMoreover,
samples. = −0.35, p <the
0.05); by of
severity
contrast, IRS1 mRNA levels were not significantly correlated with the severity of any of the
lobular inflammation was negatively correlated with hepatic IRS2 mRNA levels (r = −0.35, p < 0.05); by
Int. J. Mol. Sci. 2020, 21, 3863 4 of 17

contrast, IRS1 mRNA levels were not significantly correlated with the severity of any of the histological
features examined [26]. Enooku et al. examined 146 biopsy-proven NAFLD samples and revealed
that IRS1 mRNA levels decreased with increasing degrees of hepatic necroinflammatory activity;
however, IRS2 mRNA levels were not significantly correlated with this activity [27]. To date, the effects
of changes in hepatic IRS1/IRS2 mRNA levels on hepatic inflammation in NAFLD patients have not
been resolved.

3. Genetic Factors Affecting IR and NAFLD

To date, at least five variants in different genes have been found to be strongly associated with the
susceptibility to and progression of NAFLD, namely, PNPLA3, transmembrane 6 superfamily member
2 (TM6SF2), glucokinase regulator (GCKR), membrane bound O-acyltransferase domain-containing
7 (MBOAT7), and hydroxysteroid 17β-dehydrogenase (HSD17B13) [10]. First, the rs738409 C>G
SNP, which results in the I148M protein variant of PNPLA3, has been linked to higher hepatic fat
content but with no major effects on IR and adiposity features [10,28]. Second, the rs58542926 C>T
SNP, which encodes for the E167K variant TM6SF2, shows higher hepatic and adipose tissue IR and
enhanced muscle insulin sensitivity compared to CC homozygotes [29]. Third, the NAFLD risk variant
GCKR (P446L) is associated with higher levels of plasma low-density lipoprotein cholesterol and
triglycerides and lower fasting glucose and homeostasis model assessment parameter (HOMA)-IR [30].
Fourth, MBOAT7 suppression by obesity or the MBOAT7 rs641738 variant promotes NAFLD and
IR progression [31]. Downregulation of MBOAT7 by hyperinsulinemia contributes to fatty liver
independently of genetic background [32]. The rs626283 polymorphism in MBOAT7 is associated with
NAFLD and impaired insulin sensitivity in obese children and adolescents [33]. Finally, the rs72613567
variant in HSD17B13 plays a role in regulating retinoic acid metabolism, suggesting that retinol may
be involved in NAFLD development [10].

4. Molecular Mechanism of Hepatic IR Affects Hepatic Fibrosis

Fibrosis is the result of excessive production of extracellular matrix (ECM) that is not adequately
maintained and, thus, results in net accumulation. In the liver, hepatic stellate cells (HSCs) constitute
the main source of ECM-producing fibroblasts in models of toxic and biliary liver disease and
NAFLD [19,34]. IR is recognized as an integral component of NAFLD pathogenesis that worsens with
disease progression [19–21]. Our review suggests that the activation of HSC by IR is largely divided
into distinct direct and indirect pathways.

4.1. Indirect Pathway

Inflammation can be induced by IR itself. Hepatocyte stress and death also promote inflammation,
leading to the recruitment of macrophages and secretion of profibrogenic mediators such as transforming
growth factor-β (TGF-β), which is the center of the fibrogenic response in NASH [19,20,34,35].
There is also strong evidence for HSC activation through the direct interactions of stressed or dead
hepatocytes with HSCs. This may be through the release of profibrogenic damage-associated molecular
patterns [36,37] or other profibrogenic mediators such as Hh ligands and osteopontin (OPN) [19],
or via apoptotic bodies [38,39] that may directly act on HSCs. High mobility group box 1 released by
necrotic hepatocytes also mediates the recruitment of neutrophils in an acetaminophen-induced liver
injury model through an interaction with a receptor for advanced glycation end products [40]. C–C
chemokine receptors type 2 (CCR2) and 5 (CCR5) and their ligands CCL2 and CCL5 are implicated in
the pathogenesis of liver inflammation and fibrosis, especially in NASH [35]. In response to hepatocyte
injury, Kupffer cells secrete CCL2, which recruits monocytes to the liver. Inhibition of CCL2/CCR2 or
CCL5/CCR5 has been shown to attenuate liver fibrosis in mice [41–43], and a recent phase 2b clinical
trial showed that treatment with the CCR2/5 antagonist cenicriviroc resulted in an early antifibrotic
benefit that was maintained particularly in the subset of NASH patients with advanced fibrosis [44].
Transcriptional coactivator with PDZ binding motif (TAZ) is a paralogue of YAP and key component
Int. J. Mol. Sci. 2020, 21, 3863 5 of 17

of the HIPPO-YAP/TAZ-TAZ/TEA domain (TEAD) signaling cascade that is strongly upregulated in

hepatocytes in both mouse models and NASH patients [45]. However, TAZ was not upregulated in
simple steatosis, suggesting that TAZ could be involved in the transition from simple steatosis to NASH.
Hepatocyte TAZ to NASH fibrosis is a TEAD-mediated induction of the secretory factor Indian hedgehog
that activates fibrogenic genes in HSCs [45]. The Notch signaling pathway is important for multiple
cell differentiation processes during embryonic and adult stages [19]. Notch activity is substantially
increased in murine and human NASH [46]. In addition, hepatocyte Notch activation induces
Sox9-dependent OPN secretion, which can directly activate HSCs, independent of hepatocellular
injury, leading to collagen deposition [46]. Notch activation also increases FoxO1 activation at
gluconeogenic promoters, leading to glucose intolerance [47]. The pharmacological blockade of
Notch signaling by γ-secretase inhibitors improves glucose tolerance and IR [48]. Dongiovanni et al.
reported that IR promotes ECM stabilization by a mechanism encompassing overexpression of lysyl
oxidase-like 2 (LOXL2) in response to lipotoxicity [49]. Importantly, hepatic LOXL2 upregulation was
specifically detected in NAFLD patients with T2DM progressing to advanced fibrosis [49]. LOXL2
may be a new therapeutic target in chronic liver diseases [50]. Adiponectin, an adipokine that
modulates several metabolic processes including glucose metabolism and fatty acid oxidation, acts as
an anti-inflammatory cytokine and inhibits the development of IR and NAFLD [51,52]. Adiponectin
inhibits the differentiation of myeloid progenitor cells and modulates Kupffer cell function via reduction
of Toll-like receptor 4 signaling [52]. Adiponectin inhibits the production of inflammatory cytokines
such as TNF-α, monocyte chemotactic protein-1, and IL-6, and suppresses proinflammatory classically
activated (M1) macrophage activation [51,52]. In addition, adiponectin upregulates the production of
the anti-inflammatory cytokine IL-10 in macrophage and promotes anti-inflammatory alternatively
activated (M2) macrophage proliferation [51,52]. The indirect pathways regulating HSC activation in
steatohepatitis are summarized in Figure 2.
Int. J. Mol. Sci. 2019, 20, x FOR PEER REVIEW 5 of 18

Figure
Figure 2. The
2. The indirect
indirect pathwaysregulating
pathways regulating hepatic
hepatic stellate
stellatecell
cell(HSC)
(HSC) activation in steatohepatitis.
activation in steatohepatitis.
Metabolic
Metabolic insults
insults suchsuch as hyperinsulinemia/hyperglycemia lead
as hyperinsulinemia/hyperglycemia leadtotothe activation
the of transcriptional
activation of transcriptional
coactivator
coactivator withwith PDZ-binding
PDZ-binding motif
motif (TAZ)
(TAZ) inin hepatocytes.Increased
hepatocytes. Increased hepatocyte
hepatocyteexpression
expression of of
TAZTAZ in
in nonalcoholic steatohepatitis (NASH) but not simple steatosis directly leads to HSC activation
through the release of Indian hedgehog (Ihh) and promotes hepatocyte injury and inflammation that
may indirectly promote HSC activation. Notch activation by cell-surface ligands on a neighboring cell
leads to a Sox9-dependent increase in osteopontin (OPN) secretion to activate HSCs. HSC activation
occurs through direct interactions between stressed or dead hepatocytes (i.e., apoptosis, necrosis, or
necroptosis) and HSCs. This may be through the release of profibrogenic damage-associated
molecular patterns. High mobility group box 1 (HMGB1) is released by injured hepatocytes to
Int. J. Mol. Sci. 2020, 21, 3863 6 of 17

nonalcoholic steatohepatitis (NASH) but not simple steatosis directly leads to HSC activation through
the release of Indian hedgehog (Ihh) and promotes hepatocyte injury and inflammation that may
indirectly promote HSC activation. Notch activation by cell-surface ligands on a neighboring cell
leads to a Sox9-dependent increase in osteopontin (OPN) secretion to activate HSCs. HSC activation
occurs through direct interactions between stressed or dead hepatocytes (i.e., apoptosis, necrosis,
or necroptosis) and HSCs. This may be through the release of profibrogenic damage-associated
molecular patterns. High mobility group box 1 (HMGB1) is released by injured hepatocytes to mediate
the recruitment of neutrophils. Monocyte infiltration into the liver is primarily controlled by C–C
chemokine receptors (CCR2) and its ligand CCL2, which may serve as therapeutic targets in NASH.
Adiponectin modulates Kupffer cell function via reduction of Toll-like receptor 4 (TLR4) signaling,
and directly stimulates M1→M2 polarization.

4.2. Direct Pathway

A small number of reports suggest that hyperinsulinemia and hyperglycemia directly activate
HSCs. A study showed that IR-related hyperinsulinemia can directly stimulate HSCs to proliferate and
secrete type I collagen by differentially activating PI3K and ERK-dependent pathways [53]. Ota et al.
reported that a high-fat diet induced IR and increased the expression of the profibrotic TGF-β1 in obese
rats [54]. Recently, another report suggested that insulin-like growth factor 1 (IGF1) binds its receptor
(IGF1R) in HSCs to promote IGF1R via IRS2 to trigger ERK1/2 phosphorylation, which leads to the
expression of matrix metalloproteinase (MMP) 9 [55].
High serum glucose during hyperglycemia can lead to the activation of the HSCs [56]. Kiss et al.
reported that high glucose exposure to LX-2 cells (e.g., an immortalized human HSC cell lineage) resulted
in decreased MMP2 activity and deceleration of type I collagen in the ER, with decreased pS6 expression
pointing to development of ER stress [57]. Hyperglycemia can also aggravate hepatic fibrosis, which may
be associated with HSC autophagy induced by acid-sensing ion channel 1a, which is a subfamily of the
degenerin/epithelial Na+ channel family of the non-voltage-gated cation channel that acts through the
CaMKKβ/ERK signal pathway [58]. Although it remains unclear whether HSCs are directly activated in
prediabetic status, further study will be needed because many NAFLD patients are prediabetes. The direct
pathways regulating
Int. J. Mol. HSC
Sci. 2019, 20, activation
x FOR in steatohepatitis are summarized in Figure 3.
PEER REVIEW 7 of 18

Figure
Figure 3. The
3. The direct
direct pathways
pathways regulatingHSC
regulating HSCactivation
activation in
in steatohepatitis.
steatohepatitis.Hyperinsulinemia
Hyperinsulinemiacancan
directly stimulate HSCs to proliferate and secrete type I collagen by differentially activating PI3K-
and ERK-dependent pathways. IGF1R triggers ERK1/2 phosphorylation via IRS2, leading to the
expression of matrix metalloproteinase (MMP)-9. Hyperglycemia can aggravate hepatic fibrosis,
which may be associated with the HSC autophagy induced by acid-sensing ion channel 1a (ASIC1a).

5. Relationship Between T2DM and Hepatic Fibrosis in Patients with NAFLD

Int. J. Mol. Sci. 2020, 21, 3863 7 of 17

directly stimulate HSCs to proliferate and secrete type I collagen by differentially activating PI3K- and
ERK-dependent pathways. IGF1R triggers ERK1/2 phosphorylation via IRS2, leading to the expression
of matrix metalloproteinase (MMP)-9. Hyperglycemia can aggravate hepatic fibrosis, which may be
associated with the HSC autophagy induced by acid-sensing ion channel 1a (ASIC1a).

5. Relationship Between T2DM and Hepatic Fibrosis in Patients with NAFLD

5.1. Liver Biopsy

The gold standard for diagnosing NASH remains a liver biopsy [1]. Many past studies have
confirmed that the presence of T2DM is an independent predictor of advanced hepatic fibrosis (F3 or
F4) in biopsy-diagnosed patients with NAFLD [1–4,8,16–18]. For example, we previously reported that
T2DM was an independent predictor of advanced fibrosis in patients with NAFLD (Odds ratio (OR)
2.9, 95% confidence interval (CI) 1.3–6.1, p = 0.007) [59]. An earlier study from JSG-NAFLD showed
that in 1365 biopsy-proven Japanese NAFLD patients, 47.3% had T2DM, whereas the multivariate
analysis revealed that the presence of T2DM was one of the risk factors for advanced fibrosis (OR 2.39,
95% CI 1.60–3.55, p < 0.001) [60]. A report from Hong Kong showed that in 94 patients with T2DM who
underwent liver biopsy, 56% had steatohepatitis and 50% had advanced hepatic fibrosis [61]. A more
recent, prospective multicenter study showed that in a cohort of 458 NAFLD patients with advanced
fibrosis, 67% had T2DM, suggesting that T2DM is a robust predictor of poor transplantation-free
survival (hazard ratio (HR) 3.33) and liver-related outcomes [8]. With the increase in the number of
clinical trials, the number of observational studies of biopsy-proven NAFLD has decreased.

5.2. Serum Biomarkers

Several noninvasive biomarkers have been proposed for NASH detection and to avoid redundant
liver biopsies. In western countries, serum biomarkers for staging fibrosis include predictive models
(e.g., NAFLD fibrosis score (NFS) [62]) and direct measures of fibrosis (e.g., PIIINP [63] or Pro-C3 [64])
to discriminate between patients with advanced fibrosis [65]. Some of these markers were originally
designed for hepatitis C using the aspartate transaminase (AST)/alanine transaminase ratio, the aspartate
transaminase-to-platelet ratio index [66], and Fibrosis-4 (FIB-4) [67]. Yoneda et al. from JSG-NAFLD
reported that the platelet (PLT) count is the simplest index for predicting advanced fibrosis in patients
with NAFLD [68]. Indeed, PLT levels may be unexpectedly high even when hepatic fibrosis is advanced;
for example, patients with stage 3 hepatic fibrosis had a PLT of 189 × 109 /L, whereas patients with
stage 4 had 153 × 109 /L [68].
In Japan, fibrosis markers have also been extensively examined, including hyaluronic acid, type
IV collagen 7S, procollagen III peptide, and Wisteria floribunda agglutinin-positive Mac-2-binding
protein (WFA+ -M2BP). WFA+ -M2BP is a novel serum fibrosis biomarker for chronic hepatitis C that
is clinically validated and covered by the Japanese health insurance system [69]. WFA+ -M2BP is
also used for assessing liver fibrosis in patients with NAFLD [70,71]. Kamada et al. reported that
serum levels of Mac-2 binding protein (Mac-2bp) can be used to predict the histologic severity of
hepatic fibrosis in patients with NAFLD [72,73]. Furthermore, they tried a head-to-head comparison
between WFA+ -M2BP and Mac-2bp in which 510 patients with NAFLD from JSG-NAFLD were used.
WFA+ -M2BP and Mac-2bp were equally useful for NASH diagnosis, although Mac-2bp was superior
to WFA+ -M2BP for the prediction of the NAFLD fibrosis stage, especially for early stages fibrosis (F1
and F2) [74]. The measurement of autotaxin (ATX) has been covered by the national health insurance
in Japan since June 2018 as an auxiliary method of quantifying the degree of liver fibrosis in patients
with chronic liver disease and cirrhosis [75]. Some groups found that the serum ATX concentration
significantly correlates with the fibrosis stage in NAFLD patients [75–77]. Recently, Okanoue et al.
suggested that the combination of type IV collagen 7S and AST (CA index-NASH = 0.994×type IV
collagen 7S + 0.0255×AST) is a reliable and simple scoring system to diagnose advanced fibrosis.
The area under the receiver operating characteristics (AUROCs) for training/validation data sets are
0.842/0.931 for NASH-related advanced fibrosis [78].
Int. J. Mol. Sci. 2020, 21, 3863 8 of 17

The NFS and FIB-4 have been externally validated in populations of different ethnicities with
consistent results. Singh et al. investigated a total of 1319 patients with T2DM who underwent liver
biopsy for suspected NAFLD [79]. The diagnostic abilities were FIB-4 > 2.67 and NFS > 0.676 advanced
fibrosis, indicating reasonable specificities of 69.9% and 93% but poor sensitivities of 6.7% and 44.1%,
respectively. The AUROCs used to detect advanced fibrosis were 0.77 and 0.72, respectively. Another
report from Turkey examined 349 patients with biopsy-proven NAFLD (166 with T2DM) and showed
that a FIB-4 with a low cutoff value of 1.3 had a specificity of 67% in patients with T2DM and 69%
in those without [80]. Conversely, a FIB-4 with a high cutoff value of 2.67 had a sensitivity of 22%
in patients with T2DM and 0% in those without [80]. NFS performed similar to FIB-4, suggesting
that both FIB-4 and NFS have limited utility in diagnosing advanced fibrosis in NAFLD, especially in
patients with T2DM.

5.3. Vibration-Controlled Transient Elastography

There have been some attempts to estimate the prevalence of NASH by noninvasive methods [1].
Vibration-controlled transient elastography (VCTE) is a rapid, safe, and reproducible procedure for
liver stiffness measurement (LSM) that can be performed at the bedside with immediate results [81].
VCTE generally provides an accurate per-protocol risk assessment of advanced fibrosis (F3 or F4) in
NAFLD (AUROCs ranging from 0.80 to 0.94) [65,82,83]. Several meta-analyses, mostly performed in
viral hepatitis patients, reported good (88–89%) and excellent (93–96%) TE accuracy for diagnosing
advanced fibrosis and cirrhosis, respectively [65,84]. A recent large prospective diagnostic study
from the United Kingdom demonstrated that LSM identified patients with fibrosis from AUROCs of
0.77 (95% CI 0.72–0.82) for ≥ F2, 0.80 (95% CI 0.75–0.84) for ≥ F3, and 0.89 (95% CI 0.84–0.93) for F4.
The Youden cutoff values for F2, F3, and F4 were 8.2, 9.7, and 13.6 kPa, respectively [85]. Probe type (M
or XL probe) did not affect LSM; Oeda et al. also confirmed that liver fibrosis and steatosis could be
equally evaluated with M and XL probes in patients with NAFLD [86]. Roulot et al. examined 705
French diabetic patients and reported that 12.7% (N = 85) had LSM ≥ 8 kPa, which was suggestive of
significant fibrosis, whereas 2.1% (N = 14) had LSM ≥ 13 kPa, which indicated cirrhosis [87]. In the
Rotterdam study, a population-based study among individuals ≥ 45 y combined the presence of T2DM
and hepatic steatosis (OR, 5.20; 95% CI: 3.01–8.98; p < 0.001 for combined presence), which were
associated with LSM ≥ 8.0 kPa in multivariable analyses [88]. These studies clearly demonstrated that
the presence of T2DM, especially that concurrent with steatosis, resulted in increased probabilities of
having clinically relevant fibrosis.

5.4. Magnetic Resonance Elastography

Magnetic resonance elastography (MRE) is not operator-dependent or affected by obesity or
ascites. It is currently the most accurate imaging tool for the detection of liver fibrosis because it
is more effective than TE alone or TE with serum biomarkers combined [65,89,90]. For example,
Imajo et al. examined 142 patients with biopsy-proven NAFLD in Japan who had an average body
mass index (BMI) of 28.1 kg/m2 and underwent VCTE using M-probe, as well as MRE. The authors
found that MRE was better than VCTE for detecting fibrosis [89]. Doycheva et al. performed a
cross-sectional analysis of 100 patients with T2DM and reported that the prevalence of NAFLD
defined as MRI- proton-density fat traction ≥ 5% and advanced fibrosis defined as MRE ≥ 3.6 kPa
was 65% and 7.1%, respectively [91]. A recent study using MRE to evaluate fibrosis revealed that the
prevalence of F2 (≥ 3.0 kPa) and F3 (≥ 3.6 kPa) in the overall cohort was 5.1% and 1.3%, respectively [92].
Additionally, the prevalence of F2 and F3 in participants with NAFLD plus T2DM was 24.1% and
6.0%, respectively. In a multivariate analysis of this cohort, only age, insulin, T2DM, and fatty liver
on MR were independently associated with significant fibrosis [92]. Limitations of MRE include
cost and availability, along with patient-dependent factors such as the presence of magnetically
susceptible implants, being able to hold their breath, and claustrophobia [81]. Iron overload, high BMI,
and significant ascites were also associated with technical failure [81,93]. The benefit of MRE is that it
Int. J. Mol. Sci. 2020, 21, 3863 9 of 17

allows for a much larger sampling compared to US techniques and liver biopsy, which may reduce
sampling variability secondary to heterogeneity of fibrosis [94]. The utility of MRE in NAFLD patients
is promising, but further validation is required [65,95].

6. The Role of IR in Hepatic Fibrosis in NAFLD Patients

In 2014, Jung et al. reported that the value of the HOMA-IR was significantly higher in NASH
subjects than in healthy controls (4.4 ± 2.5 vs. 1.7 ± 0.6; p < 0.001), but that this was not the case in patients
with T2DM [96]. Unfortunately, their study could not show a significant correlation of HOMA-IR
to the severity of either histologic grading or staging, because of the small sample size (41 cases of
biopsy-proven NAFLD). Kessoku et al. evaluated 1365 biopsy-proven NAFLD patients registered
in the JSG-NAFLD database and showed that HOMA-IR significantly increased depending on the
degree of hepatic fibrosis (2.7 ± 1.1 in stage 0, 3.5 ± 1.6 in stage 1, 4.0 ± 1.7 in stage 2, 4.3 ± 1.7 in stage 3,
and 4.6 ± 2.1 in stage 4; p < 0.01) [97]. Ballestri et al. in Italy investigated 118 consecutive biopsy-proven
NAFLD patients (25% with T2DM) and reported that HOMA-IR independently predicted advanced
hepatic fibrosis [98]. In both studies, the subjects included patients with T2DM. In fact, the prevalence
of T2DM among patients with NAFLD has been reported to be 22.51% in global studies [4]. Conversely,
approximately 80% of NAFLD patients are nondiabetic; therefore, can HOMA-IR also act as an
independent predictor of advanced fibrosis even in nondiabetic patients? We recently investigated
361 biopsy-proven Japanese NAFLD patients without T2DM who were registered with JSG-NAFLD.
We reported that HOMA-IR ≥ 2.90 was an independent predictor of advanced fibrosis in nondiabetic
NAFLD patients, and our data suggest that there may be a pathway for IR to directly activate HSCs [99].
Unfortunately, there are no US Food and Drug Administration-approved medications to treat NAFLD
and guidelines for NAFLD management are not well established. In this context, improving IR may be
beneficial for anti-fibrotic treatment, even among patients with nondiabetic NAFLD [100,101].

7. Hepatocellular Carcinoma
The exact pathogenesis of HCC in NAFLD has not been fully described; however, both obesity
and T2DM seem to play a critical role in hepatocarcinogenesis [102,103]. The global annual incidence
of HCC in people with NAFLD is estimated to be 0.44 per 1000 person-years, whereas for those with
NASH, the incidence of HCC is higher at 5.29 per 1000 person-years, which most likely results from the
inflammatory state in those with NASH that promotes fibrosis and disease progression [4]. Tokushige
et al. from JSG-NAFLD examined 532 patients with alcoholic liver disease (ALD)-HCC and 209 patients
with NAFLD-HCC and revealed that the prevalence of lifestyle-related diseases including T2DM and
hypertension was higher in the NAFLD-HCC group than in the ALD-HCC group [104]. In one study
using data from the Surveillance, Epidemiology, and End Results database that covered a six-year
period of time from 2004 to 2009, investigators determined that there was a 9% annual increase in the
number of HCC cases attributed to NAFLD [105]. In another study using data from four European
primary care databases representing the UK, Netherlands, Italy, and Spain, the authors reported that
coded NAFLD/NASH patients were more likely to have T2DM, hypertension, and obesity than the
matched controls [106]. The HR for HCC in patients compared to those in controls was 3.51 (95%
CI 1.72–7.16). The strongest independent predictor of an HCC or cirrhosis diagnosis was baseline
diagnosis of T2DM [106]. A recent study demonstrated with a multivariate analysis of 354 patients
with NASH cirrhosis that T2DM was associated with an increased risk of developing HCC (HR 4.2;
95% CI 1.2–14.2) [107]. Although the risk of HCC is higher in NASH-related cirrhosis, there is also
evidence that HCC can occur in the absence of cirrhosis (26–37%) in both NAFLD and NASH [108–110].
Bengtsson et al. examined the mortality rate and concluded that the parameters that independently
associate with increased mortality included the Barcelona Clinic Liver Cancer stage, number of tumors,
lower albumin, and presence of T2DM [110].
A number of genetic factors have also been implicated in the development of NAFLD. PNPLA3
polymorphisms were noted to not only lead to an increased risk of steatohepatitis and fibrosis, but also
Int. J. Mol. Sci. 2020, 21, 3863 10 of 17

of a three-fold increased risk of HCC. These risks were independent of age, gender, BMI, T2DM,
and presence of fibrosis or cirrhosis [103]. However, whether carrying the transmembrane 6 superfamily
member 2 (TM6SF2; rs58542926 c.449 C>T, p.E167K) polymorphism is an independent HCC risk factor
is still controversial [103,111,112].

8. Conclusions
In this article, several aspects that potentially contribute to the mechanisms underlying IR and
T2DM in the progression of NAFLD are discussed. We also discussed the indirect/direct roles of IR
during the progression of hepatic fibrosis. More detailed knowledge of these mechanisms in NAFLD
are needed to identify novel therapeutic approaches for this disease. Furthermore, specific biomarkers
that can predict the degree of liver damage and NAFLD progression to cirrhosis and cancer should be
developed. To achieve these objectives, we need animal models that accurately reflect the metabolic and
histological characteristics of human NAFLD. Further investigations using such models can provide
additional insight into the underlying mechanism by which extrahepatic organs (e.g., gut, adipose
tissue, brain) regulate IR.

Author Contributions: Conceptualization, H.F.; original draft preparation, H.F.; review and editing, H.F. and
N.K.; supervision: N.K. All authors have read and agreed to the final version of the manuscript.
Funding: This work was supported by a Grant-in-Aid for Scientific Research (C) from the Japan Society for the
Promotion of Science (grant number 17K09437) to H.F. (2017–2020).
Acknowledgments: The authors are grateful to M. Sato-Matsubara for useful discussions. We acknowledge
the scientific contribution and support of the JSG-NAFLD in the Japan Strategic Medical Administration
Research Center (J-SMARC). Division of Hepatology and Pancreatology, Department of Internal Medicine, Aichi
Medical University (Nagakute, Japan): Yoshio, Sumida, Satoshi Kimoto, and Masashi, Yoneda. Department of
Gastroenterology and Hepatology, Yokohama City University Graduate School of Medicine (Yokohama, Japan):
Masato, Yoneda, Kento Imajo, Yuji Ogawa, Yasushi Honda, Takaomi Kessoku, and Atsushi Nakajima. Department
of General Internal Medicine2, Kawasaki Medical School (Okayama, Japan): Miwa Kawanaka. Liver Center, Saga
University Hospital (Saga, Japan): Hirokazu Takahashi. Eguchi Hospital (Saga, Japan): Yuichiro Eguchi. Division
of Gastroenterology and Hepatology, Department of Internal Medicine, Tokyo Women’s Medical University
Medical Center East (Tokyo, Japan): Masafumi Ono. Department of Gastroenterology, National Center for
Global Health and Medicine (Tokyo, Japan): Yuichi Nozaki. Division of Cardiovascular Medicine, Department
of Medicine, Osaka University Graduate School of Medicine (Osaka, Japan): Masahiro Koseki. Department
of Gastroenterology and Hepatology, Suita Municipal Hospital (Osaka, Japan): Yuichi Yoshida. Division of
Gastroenterology, Department of Medicine, Kurume University School of Medicine (Kurume, Japan): Takumi
Kawaguchi. Department of Molecular Biochemistry & Clinical Investigation, Osaka University Graduate School
of Medicine (Osaka, Japan): Yoshihiro Kamada. Hepatology Center, Saiseikai Suita Hospital (Osaka, Japan):
Takeshi Okanoue. Department of Hepatology, Gifu Municipal Hospital (Gifu, Japan): Hideki Hayashi, Yoichi
Nishigaki, Takafumi Uchiki, Yusuke Suzuki and Eiichi Tomita. Department of Internal Medicine, Himeji Red Cross
Hospital (Himeji, Japan): Toshifumi Tada. Department of Gastroenterology and Metabolism, Graduate School of
Biomedical and Health Sciences, Hiroshima University (Hiroshima, Japan): Takashi Nakahara. Department of
Internal Medicine II, Shimane University Faculty of Medicine (Izumo, Japan): Hiroshi Tobita and Shuichi Satoh.
Department of Medicine, Division of Gastroenterology and Hematology/Oncology, Asahikawa Medical University
(Asahikawa, Japan): Koji Sawada. Department of Gastroenterology and Hepatology, Kochi Medical School (Kochi,
Japan): Kensuke Munekage and Tsunehiro Ochi. Department of Internal Medicine, Institute of Gastroenterology,
Tokyo Women’s Medical University (Tokyo, Japan): Tomomi Kogiso, and Katsutoshi Tokushige. Department of
Gastroenterology and Neurology, Faculty of Medicine, Kagawa University (Kagawa, Japan): Tomohiro Morishita.
Department of Gastroenterology, Fukushima Medical University School of Medicine (Fukushima, Japan): Atsushi
Takahashi. Second Department of Internal Medicine, Osaka Medical College (Osaka, Japan): Shinya Fukunishi.
Department of Hepatology, Hamamatsu University School of Medicine (Hamamatsu, Japan): Kazuhito Kawata.
Department of General Internal Medicine, Fukui-ken Saiseikai Hospital (Fukui, Japan): Kazuo Notsumata.
Division of Gastroenterology and Hepatology, Department of Internal Medicine, National Defense Medical College
(Tokorozawa, Japan): Kengo Tomita. Department of Liver, Biliary Tract and Pancreas Diseases, Fujita Health
University (Toyoake, Japan): Naoto Kawabe. Department of Gastroenterology, JA Hiroshima General Hospital
(Hiroshima, Japan): Michihiro Nonaka, and Hideyuki Hyogo. Division of Gastroenterology, Department of Internal
Medicine, Nippon Medical School Musashi Kosugi Hospital (Kawasaki, Japan): Taeang Arai. Tokyo Medical
University Ibaraki Medical Center, Ibaraki, Japan: Tadashi Ikegami. Department of Gastroenterology and
Hepatology, Ogaki Municipal Hospital (Ogaki, Japan): Hidenori Toyoda and Takashi Kumada.
Conflicts of Interest: The authors declare that they have no conflicts of interest.
Int. J. Mol. Sci. 2020, 21, 3863 11 of 17

Abbreviations
AKT Protein kinase B
ALD Alcoholic liver disease
AST Aspartate transaminase
ATX Autotaxin
AUROC Area under the receiver operating characteristic
BMI Body mass index
CCR C-C chemokine receptors
CI Confidence interval
ECM Extracellular matrix
ER Endoplasmic reticulum
ERK RAS extracellular signal-regulated kinase
GCK Glucokinase regulator
HCC Hepatocellular carcinoma
HOMA Homeostasis model assessment parameter
HR Hazard ratio
HSC Hepatic stellate cell
HSD17B13 Hydroxysteroid 17β-dehydrogenase
IGF Insulin-like growth factor
IGN Intestinal gluconeogenesis
IKK Inhibitor of nuclear factor-κB kinase
IL Interleukin
IR Insulin resistance
IRS Insulin receptor substrate
JNK c-Jun N-terminal kinase
JSG-NAFLD Japan Study Group of NAFLD
LOXL2 Lysyl oxidase-like 2
LSM Liver stiffness measurement
Mac-2bp Mac-2 binding protein
MBOAT7 Membrane bound O-acyltransferase domain- containing 7
MMP Matrix metalloproteinase
MRE Magnetic resonance elastography
NAFLD Nonalcoholic fatty liver disease
NASH Nonalcoholic steatohepatitis
NF-κB Nuclear factor-kappa B
NFS NAFLD fibrosis score
OPN Osteopontin
OR Odds ratio
PDK Phosphoinositide-dependent kinase
PI3K Phosphatidylinositol 3-kinase
PNPLA3 Patatin-like phospholipase domain containing 3
SOCS Suppressor of cytokine signaling
TAZ Transcriptional coactivator with PDZ- binding motif
TEAD TAZ/TEA domain
TGF-β Transforming growth factor-β
T2DM Type2 diabetes mellitus
TM6SF2 Transmembrane 6 superfamily member 2
TNF Tumor necrosis factor
VCTE Vibration-controlled transient elastography
WFA+ -M2bp Wisteria floribunda agglutinin-positive Mac-2-binding protein
Int. J. Mol. Sci. 2020, 21, 3863 12 of 17

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