Journal of

Clinical Medicine

Review
Biomarkers for Assessing Non-Alcoholic Fatty Liver Disease in
Patients with Type 2 Diabetes Mellitus on Sodium–Glucose
Cotransporter 2 Inhibitor Therapy
Farah Khaznadar 1,2 , Ana Petrovic 1 , Omar Khaznadar 3 , Hrvoje Roguljic 1,2,4 , Kristina Bojanic 1,2,5 ,
Lucija Kuna Roguljic 1 , Stjepan Siber 1 , Robert Smolic 1 , Ines Bilic-Curcic 2,4 , George Y. Wu 6
and Martina Smolic 1, *

1 Faculty of Dental Medicine and Health Osijek, Josip Juraj Strossmayer University of Osijek,
31000 Osijek, Croatia; [Link].8@[Link] (F.K.); anapetrovic@[Link] (A.P.);
hroguljic@[Link] (H.R.); kbojanic@[Link] (K.B.); lkuna@[Link] (L.K.R.); ssiber@[Link] (S.S.);
rsmolic@[Link] (R.S.)
2 Faculty of Medicine Osijek, Josip Juraj Strossmayer University of Osijek, 31000 Osijek, Croatia;
ibcurcic@[Link]
3 Department of Radiology, “Dr. Juraj Njavro” National Memorial Hospital Vukovar, 32000 Vukovar, Croatia;
[Link]@[Link]
4 Clinical Hospital Center, 31000 Osijek, Croatia
5 Health Center Osijek-Baranja County, 31000 Osijek, Croatia
6 Department of Medicine, Division of Gastrenterology/Hepatology, University of Connecticut Health Center,
Farmington, CT 06030, USA; wu@[Link]
* Correspondence: msmolic@[Link]

Abstract: In the current modern era of unhealthy lifestyles, non-alcoholic fatty liver disease (NAFLD)
is the most prevalent liver disease and has become a serious global health problem. To date, there is
no approved pharmacotherapy for the treatment of NAFLD, and necessary lifestyle changes such as
Citation: Khaznadar, F.; Petrovic, A.;
Khaznadar, O.; Roguljic, H.; Bojanic,
weight loss, diet, and exercise are usually not sufficient to manage this disease. Patients with type
K.; Kuna Roguljic, L.; Siber, S.; Smolic, 2 diabetes mellitus (T2DM) have a significantly higher risk of developing NAFLD and vice versa.
R.; Bilic-Curcic, I.; Wu, G.Y.; et al. Sodium–glucose cotransporter 2 (SGLT2) inhibitors are antidiabetic agents that have recently been
Biomarkers for Assessing approved for two other indications: chronic kidney disease and heart failure in diabetics and non-
Non-Alcoholic Fatty Liver Disease in diabetics. They are also emerging as promising new agents for NAFLD treatment, as they have shown
Patients with Type 2 Diabetes beneficial effects on hepatic inflammation, steatosis, and fibrosis. Studies in animals have reported
Mellitus on Sodium–Glucose favorable effects of SGLT2 inhibitors, and studies in patients also found positive effects on body mass
Cotransporter 2 Inhibitor Therapy. J. index (BMI), insulin resistance, glucose levels, liver enzymes, apoptosis, and transcription factors.
Clin. Med. 2023, 12, 6561. https://
There are some theories regarding how SGLT2 inhibitors affect the liver, but the exact mechanism
[Link]/10.3390/jcm12206561
is not yet fully understood. Therefore, biomarkers to evaluate underlying mechanisms of action of
Academic Editor: Gian SGLT2 inhibitors on the liver have now been scrutinized to assess their potential as a future in-label
Paolo Caviglia therapy for NAFLD. In addition, finding suitable non-invasive biomarkers could be helpful in clinical
practice for the early detection of NAFLD in patients. This is crucial for a positive disease outcome.
Received: 15 September 2023
Revised: 11 October 2023
The aim of this review is to provide an overview of the most recent findings on the effects of SGLT2
Accepted: 13 October 2023 inhibitors on NAFLD biomarkers and the potential of SGLT2 inhibitors to successfully treat NAFLD.
Published: 16 October 2023
Keywords: SGLT2 inhibitors; NAFLD; biomarkers; diabetes mellitus type 2

Copyright: © 2023 by the authors.

Licensee MDPI, Basel, Switzerland.
1. Introduction
This article is an open access article
distributed under the terms and
The prevalence of non-alcoholic fatty liver disease (NAFLD), the most common liver
conditions of the Creative Commons
disease, has been reported to be as high as 25%. It is characterized by the presence of
Attribution (CC BY) license (https://
fat in at least 5% of hepatocytes [1,2]. Lipid homeostasis in the liver is controlled by a
[Link]/licenses/by/ very complex interplay between nuclear receptors, transcription factors, and hormones.
4.0/). Excessive fat storage occurs when the balance between lipogenesis/lipid uptake vs. lipid

J. Clin. Med. 2023, 12, 6561. [Link] [Link]

J. Clin. Med. 2023, 12, 6561 2 of 15

oxidation/excretion is disrupted [3,4]. NAFLD is usually associated with metabolic syn-

drome and is not, by definition, caused by excessive alcohol intake (defined as ≥20 g per
day for women and ≥30 g per day for men) [1,2]. NAFLD represents a wide range of liver
diseases, but histologically, it can be divided into two different types: non-alcoholic fatty
liver (simple steatosis or NAFL) and non-alcoholic steatohepatitis (NASH), which is the
more aggressive form [5–7]. Liver inflammation and injury are typical for NASH, and this
form of NAFLD could lead to liver cirrhosis and hepatocellular cancer [2,8]. Due to the slow
and asymptomatic progression of NAFLD, it is often diagnosed at a late stage. NAFLD
has already become the second most common indication for liver transplantation. It takes
around three to seven years for the development of NASH, which occurs in one-fifth of
patients with NAFLD [9,10]. By the year 2027, it is expected that 18 million people in
Japan, the US, and Europe (Germany, England, Italy, Spain, and France) will be affected by
NASH [2]. Genetic predisposition also plays a role in NAFLD development, and the most
common polymorphisms detected in NAFLD patients are genetic variations of transmem-
brane 6 superfamily member 2 (TM6SF2), patatin-like phospholipase domain-containing
protein 3 (PNPLA3), and glucokinase regulatory protein (GCKR) [11].
A meta-analysis revealed that patients with type 2 diabetes mellitus (T2DM) have
more than two-fold higher risk of developing serious hepatic disease [12]. Other studies
have also confirmed T2DM as a risk factor for NAFLD. Conversely, patients with NAFLD
have an increased risk of developing T2DM [13].
Currently, lifestyle modifications such as weight loss and physical activity are es-
sential for all patients with NAFLD but are mostly not successful in the management
of disease progression. Thus, there is an urgent need for approved medication therapy
for NAFLD [4,5,14,15]. Ideal pharmacotherapy for NAFLD should decrease steatosis and
inflammation, preferably liver fibrosis, while simultaneously improving insulin resistance,
adiposity, and serum glucose levels [14]. There are currently numerous agents under clini-
cal studies, and one of the leading candidates for Food and Drug Administration (FDA)
approval are sodium–glucose cotransporter 2 (SGLT2) inhibitors [2,16,17]. Canagliflozin
(Invokana), dapagliflozin, empagliflozin, ipragliflozin, tofogliflozin, and ertugliflozin, also
known as “flozins”, are glucose-lowering agents with an insulin-independent mode of
action [18–22]. They were FDA-approved as oral antidiabetic drugs to reduce glucose levels
by the inhibition of glucose reabsorption in the proximal renal tubule and are, in general,
not associated with a risk of hypoglycemia [23,24]. In general, SGLT2 inhibitors have a good
safety profile, and the most frequent side effects reported are infection of the genitourinary
tract and hypotension [16]. After gaining approval for T2DM, SGLT2 inhibitors were also
approved for chronic kidney disease and heart failure in the non-diabetic population due
to their beneficial effects on the cardiovascular and renal systems [5,25]. In general, the
antifibrotic and anti-inflammatory effects of SGLT2 inhibitors have been proposed as a com-
mon mode of action for liver, kidney, and heart protection [26]. In this review, we provided
an overview of the effects of SGLT2 inhibitors on NAFLD development with a focus on
biomarkers that could be useful for early screening of NAFLD in patients with T2DM.

Effects of SGLT2 Inhibitors on Liver

The conversion of carbohydrates from the bloodstream into fatty acids and ultimately
into triglycerides or other lipids is a highly coordinated process called de novo lipogene-
sis [27]. Catabolism and lipolysis in extrahepatic tissues, especially adipose tissue, result in
the transfer of free fatty acids to the liver, where excess fat results in liver steatosis. Insulin
regulates both de novo lipogenesis and lipolysis. Since insulin resistance is usually present
in NAFLD patients, insulin is unable to adequately inhibit adipose lipolysis [28]. Since
insulin resistance is usually present in NAFLD patients, insulin is unable to adequately
inhibit adipose lipolysis [28]. Furthermore, adipose tissue affects NAFLD progression by
producing hormones and cytokines, which contribute to the dysfunction of hepatocytes
and by increasing liver uptake of lipids [29]. Lipoprotein lipase (LPL), the enzyme that
regulates the rate of hydrolysis of VLDL and triglycerides, is also involved in NAFLD
J. Clin. Med. 2023, 12, 6561 3 of 15

development due to upregulation [4]. Dysregulation of liver lipid metabolism is associated

with metabolic diseases such as T2DM and fatty liver. Therefore, identifying biomarkers
that contribute to the impairment of this process could be important in assessing liver
steatosis [27,28].
There are several mechanisms by which SGLT2 inhibitors improve liver status and
decrease liver fat in patients with T2DM. For example, inhibition of de novo lipogenesis
by lowering glucose and insulin levels and increasing β-oxidation by elevating glucagon
levels could shift the emphasis of metabolism from carbohydrate to fatty acid [5]. Some
studies on SGLT2 inhibitor therapy in patients with or without T2DM have reported
no significant correlation between changes in body mass index (BMI) and liver steatosis
parameters, specifically the controlled attenuation parameter (CAP), an ultrasound method
for measuring hepatic fat content. This confirmed that SGLT2 inhibitors have an impact on
liver steatosis independent of weight loss [30,31]. Apart from decreasing oxidative stress
caused by abnormally high glucose levels, SGLT2 inhibitors can downregulate pro-oxidants
and free radical development and upregulate antioxidant molecules such as glutathione
peroxidases (GSHs) and superoxide dismutases (SODs) [5,32–34].
The inhibition of inflammatory molecules such as interleukin-6 (IL-6) and tumor
necrosis factor α (TNF-α) could be involved in SGLT2 inhibitors’ beneficial mechanisms in
the liver [26]. Indeed, Bellanti et al. analyzed the impact of empagliflozin, dapagliflozin,
and canagliflozin in 26 NAFLD patients and reported decreased levels of IL-6, IL-1ß, and
TNF, as well as beneficial effects on liver steatosis and fibrosis [35].
Because ketone bodies have been detected in patients with T2DM treated with da-
pagliflozin and empagliflozin, it is considered that this class of medication triggers the
disposal of fatty acids from liver lipid tissue, which are then oxidized to produce ketones
due to a decrease in insulin and an increase in glucagon [36]. Another clinical study re-
ported that ipragliflozin improved liver condition in a subgroup of 25 patients with hepatic
steatosis, independent of body weight reduction [37]. Another study suggested that the
beneficial effect of SGLT2 inhibitors on the liver triggered hepatic autophagy, a process
of self-regeneration normally occurring in the fasting state [38,39]. Studies on animal
models have shown that SGLT2 inhibitors induce autophagy by targeting the adenosine
monophosphate-activated protein kinase-mammalian/mechanistic of rapamycin (AMPK-
mTOR) signaling pathway [40,41]. It was found that during autophagy, dapagliflozin
increased lipid oxidation and levels of amino acids leucine and valine, which could be
future biomarkers of therapeutic efficacy with SGLT2 inhibitors [38].
An ongoing phase 3 clinical trial, DEAN (NCT03723252), aims to assess the efficacy
and safety of dapagliflozin in 100 participants with T2DM and NASH. Outcomes for liver
improvement and metabolic risk parameters will be studied [42,43]. Another ongoing phase
4 clinical trial (NCT05254626) aims to assess the effectiveness and safety of dapagliflozin
compared with another antidiabetic drug, pioglitazone, in NASH patients with and without
diabetes [43]. Interestingly, the recommendation to introduce SGLT2 inhibitors in NAFLD
patients with T2DM is already included in guidelines developed by the Japanese Society of
Gastroenterology in collaboration with the Japan Society of Hepatology [44].

2. Biomarkers
Microbiota and the gut–liver interaction play a very important role in liver home-
ostasis since most of the liver blood circulation comes from the gut. Leaky gut, altered
microbiome, and impaired toxin clearance have been reported to be associated with NAFLD
progression [5,9]. It is reported that patients with NAFLD consume significantly more
fructose than controls [13]; fructose from soft drinks and Ruminococcus bacteria were found
to be contributors to liver fibrosis [9]. Dapagliflozin improved microbial abundance and
variety in mice with diabetes, while in control healthy mice, only a minor effect was ob-
served, suggesting a potential beneficial effect of SGLT2 inhibitors on gut microbiota when
associated with T2DM [45]. Furthermore, in a 3-month clinical trial, empagliflozin reduced
the number of noxious bacteria Escherichia-Shigella, Hungatella, and Bilophila present in
J. Clin. Med. 2023, 12, 6561 4 of 15

the gut microbiota of patients with T2DM [46]. Furthermore, SGLT2 inhibitors have been
shown to induce increased short-chain fatty acid (SCFA)-producing bacteria, resulting in
improved amino acid metabolism [47,48]. This is relevant because increased SCFAs have
shown effects such as increased insulin sensitivity and a reduction in the hepatic fat storage
in NAFLD. However, excessive SCFAs could also inhibit hepatic AMPK in the liver and
increase the accumulation of hepatic FFAs [49,50]. Thus, in terms of microbiota and its
metabolites as biomarkers, further research is required.
Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels, due to
hepatic inflammation, and gamma-glutamyl transferase (GGT) are the most used biomark-
ers for the assessment of liver disease. However, their values should be interpreted with
caution as the patients with NAFLD can have normal levels. In addition, reduction in amino-
transferase levels is not necessarily indicative of liver histology improvement [1,30,51].
However, SGLT2 inhibitors have effects on liver enzymes, as was reported in several studies
on patients with NAFLD [20,52,53]. One prospective study in Japan on 43 T2DM patients
with NAFLD reported decreased levels of GGT, AST, and ALT after 24 weeks of therapy
with ipragliflozin [20]. Hayashi et al. assessed the effects of dapagliflozin on the lipid
profile in 40 T2DM patients. Besides the inhibition of strong atherogenic sd low-density
lipoprotein-C (sd LDL-C) and elevation of favorable high-density lipoprotein 2-C (HDL2-C),
the study found significantly decreased plasma concentrations of both ALT and AST in
dapagliflozin group 3 months after therapy initiation [52]. In another randomized clinical
trial with dapagliflozin in T2DM patients with NAFLD, reduced GGT and ALT levels in
the serum of the dapagliflozin-treated group were reported [53]. A meta-analysis and
systematic review by Amjad et al., which included therapy with different SGLT2 inhibitors
(ipragliflozin, empagliflozin, dapagliflozin, canagliflozin, and luseogliflozin) also demon-
strated that SGLT2 inhibitors significantly improve liver status by lowering AST and ALT
levels [54]. In Table 1, we provide an adapted summary of this study [54] updated with the
most recently obtained evidence, as well as a detailed investigated biomarker list and their
behavior in SGLT2 inhibitor treatment.

Table 1. Randomized controlled trials (RCT) on patients with T2DM treated with SGLT2 inhibitors
and observed effect on the biomarkers. Adapted and updated from Amjad et al. [54].

Author and Year SGLT2 Inhibitor Effect on Biomarker Investigated Main Observed Effect on Liver
Liver biopsy—reduced liver Improved liver fibrosis and reduced
Takahashi et al., 2022 [55] Ipragliflozin
fat content liver fat deposition (improved NASH)
Latva-Rasku et al., 2019 [56] Dapagliflozin FGF21 reduction Reduced liver fat deposition
MRI-assessed liver fat
content (PDFF)
Eriksson et al., 2018 [57] Dapagliflozin Reduced liver fat deposition
CK18-M65 and CK18-M30
decreased
MRI-assessed liver fat content
Kuchay et al., 2018 [58] Empagliflozin Reduced liver fat deposition
(PDFF) reduced
CT obtained liver/spleen ratio
Shibuya et al., 2017 [59] Luseogliflozin Reduced liver fat deposition
reduced
Ito et al., 2017 [60] Ipragliflozin FIB-4 index improved Improved liver fibrosis
Transient elastography
Improved liver fibrosis (preventing
Shimizu et al., 2019 [53] Dapagliflozin (FibroScan)—CAP parameters
progression)
improved
Takeshita et al., 2022 [22] Tofogliflozin FIB-4 index improved Improved liver fibrosis
Adiponectin increased
Kahl et al., 2020 [61] Empagliflozin Reduced liver fat deposition
Uric acid decreased
J. Clin. Med. 2023, 12, 6561 5 of 15

Table 1. Cont.

Author and Year SGLT2 Inhibitor Effect on Biomarker Investigated Main Observed Effect on Liver
Transient elastography
Han et al., 2020 [62] Ipragliflozin (FibroScan)—CAP parameter and Reduced liver fat deposition
FLI improved
Improved liver function: AST and
AST, ALT, HDL, and LDL ALT decreased. Suppression of potent
Hayashi et al., 2017 [52] Dapagliflozin
values decreased atherogenic sd LDL-C and increased
favorable HDL2-C
MRI-assessed liver fat content
Harreiter et al., 2021 [63] Dapagliflozin (PDFF), fatty liver index (FLI) Reduced liver fat deposition
has improved
ALT values decreased, Liver/spleen Improved liver function, reduced
Bando et al., 2017 [64] Ipragliflozin
ratio increased liver fat deposition
Kinoshita et al., 2020 [65] Dapagliflozin Liver/spleen ratio increased Reduced liver fat deposition
Transient elastography
Chehrehgosha et al., 2021 [66] Empagliflozin (FibroScan)—CAP parameters Reduced liver fat deposition
improved
MRI-assessed liver fat content
Yoneda et al., 2021 [67] Tofogliflozin Reduced liver fat deposition
(PDFF) reduced
FGF21 = Fibroblast growth factor 21, MRI = Magnetic resonance imaging, PDFF = Proton density fat fraction,
CT = Computerized tomography, CAP = Controlled attenuation parameter, AST = Aspartate aminotransferase,
ALT = Alanine aminotransferase, HDL = High-density lipoprotein, LDL = Low-density lipoprotein, FLI = Fatty
liver index.

2.1. Non-Invasive Physical Methods

The most accurate method for NAFLD diagnosis is still liver biopsy, but it is inva-
sive with potential adverse events [1,14]. As alternatives to biopsy, some non-invasive
approaches to the diagnosis have been developed. Transient elastography (TE) and
Magnetic Resonance Imaging (MRI) are the two most common techniques for NAFLD
assessment [68].
Transient elastography (TE) is an ultrasound technique that uses FibroScan to measure
at the same time two different parameters, CAP and Liver Stiffness Measurement (LSM),
utilizing the same radiofrequency information [66,69,70]. CAP is a method that measures
increased attenuation of the ultrasound beam when it crosses the liver tissue and is used
for the evaluation of liver steatosis [69,71]. LSM is used for detecting liver fibrosis by
measuring the velocity of the shear wave, which is mechanically produced when passing
through the liver tissue [72]. Transient elastography was used in a 24-week randomized
clinical trial to assess the effects of dapagliflozin on liver steatosis and fibrosis in patients
with T2DM and NAFLD by measuring CAP and LSM values [53]. In the dapagliflozin
group, the CAP value and visceral fat were significantly decreased, and the LSM value
was significantly decreased in the severe fibrosis subgroup [53]. A double-blind clinical
trial including 43 patients treated with empagliflozin and 47 with placebo demonstrated a
significant decrease in LSM in the empagliflozin group (p = 0.001) and a significant decrease
in the CAP parameter (p = 0.035) in a subgroup of patients treated with empagliflozin with
more severe steatosis at baseline (CAP > 302 dB/m) [30]. Two other studies involving
NAFLD and T2DM patients on empagliflozin therapy also reported significant decreases in
CAP and LSM values [66,71]. A recent pilot study has demonstrated that the combination
of empagliflozin and metformin compared to metformin monotherapy alleviated hepatic
steatosis to a greater extent, showing significantly lower CAP value in patients with T2DM
with dual therapy [73].
 significant decrease in the CAP parameter (p = 0.035) in a subgroup of patients treated
with empagliflozin with more severe steatosis at baseline (CAP > 302 dB/m) [30]. Two
other studies involving NAFLD and T2DM patients on empagliflozin therapy also
reported significant decreases in CAP and LSM values [66,71]. A recent pilot study has
demonstrated that the combination of empagliflozin and metformin compared to
J. Clin. Med. 2023, 12, 6561 metformin monotherapy alleviated hepatic steatosis to a greater extent, showing 6 of 15
significantly lower CAP value in patients with T2DM with dual therapy [73].

[Link]
2.2. PotentialBiomarkers
BiomarkersofofSteatosis
Steatosis
AAnumber
numberofofserological
serologicalbiomarkers
biomarkershave havebeenbeenrecommended
recommendedfor fordiagnosis
diagnosisand and
NAFLDtreatment
NAFLD treatmentand andmonitoring
monitoring[74].
[74].TheThefatty
fattyliver
liverindex
index(FLI)
(FLI)isiscalculated
calculatedbasedbasedon on
triglycerides,GGT,
triglycerides, GGT,BMI, BMI,
and and
waistwaist circumference
circumference values
values and and is a commonly
is a commonly used biomarker used
tobiomarker to predict
predict excessive excessive
liver liver fat
fat content. content.
A risk scoreAofriskFLIscore
> 60% ofisFLI > 60% isof
indicative indicative
excessiveof
hepatic
excessivefat,hepatic
while an FLI
fat, < 30%
while an rules
FLI <it30%outrules
[23,75].
it out [23,75].
Peroxisome
Peroxisomeproliferator-activated
proliferator-activatedreceptors
receptors(PPARα,
(PPARα,PPARβ/δ,
PPARβ/δ, and and PPARγ)
PPARγ)are areaa
family
family of nuclear receptors acting as transcription factors [76]. Endogenous ligandsthat
of nuclear receptors acting as transcription factors [76]. Endogenous ligands that
activate
activatePPARs
PPARsare arelipids
lipidsand
andtherefore
thereforethe thedysregulation
dysregulationofofPPARs PPARsisisassociated
associatedwith with
metabolic
metabolicdisorders,
disorders,which
which makes
makesthem themespecially
especiallysuitable diagnostic
suitable and and
diagnostic treatment tar-
treatment
gets (Figure
targets 1) [76,77].
(Figure Although
1) [76,77]. Although these
thesethree
threeisoforms
isoforms have
have different
differentroles
rolesininliver
liverlipid
lipid
metabolism,
metabolism,they theyall
allhave
havean
ananti-inflammatory
anti-inflammatoryeffect effectononthetheliver
liver[28].
[28].Studies
Studieson onpatients
patients
have suggested that PPARs are involved in NAFLD development
have suggested that PPARs are involved in NAFLD development [28]. The [28]. The downregulation
ofdownregulation
PPARγ was proposed of PPARγ as awas
mechanism
proposedof asdecreased
a mechanism liverofadipogenesis
decreased liver in NAFLD
adipogenesisin a
study on mice treated with empagliflozin [78]. In the same study,
in NAFLD in a study on mice treated with empagliflozin [78]. In the same study, empagliflozin improved
mitochondrial oxidationmitochondrial
empagliflozinβimproved via the upregulation
β oxidationof PPARα,
via the which was suppressed
upregulation of PPARα,due which to
the
washigh-fat diet [78].
suppressed due to the high-fat diet [78].

[Link]
Figure Effects of SGLT2
of SGLT2 inhibitors
inhibitors on potential
on potential NAFLD
NAFLD biomarkers.
biomarkers. SG GCKR:SG GCKR: Glucokinase
Glucokinase regula-
regulatory protein; ChREBP: Carbohydrate-responsive element-binding protein; SREBP1c:
tory protein; ChREBP: Carbohydrate-responsive element-binding protein; SREBP1c: Sterol response Sterol
response element binding protein 1c; FAS: Fatty acid synthase; MTTP: Microsomal triglyceride
element binding protein 1c; FAS: Fatty acid synthase; MTTP: Microsomal triglyceride transfer protein;
transfer protein; PPARα: Peroxisome proliferator-activated receptor α; PPARγ: Peroxisome
PPARα: Peroxisome proliferator-activated receptor α; PPARγ: Peroxisome proliferator-activated
proliferator-activated receptor γ; FA: fatty acid; TG: Triglyceride; FGF21: Fibroblast growth factor
receptor
21; ALT:γ; FA: fatty acid;
Alanine TG: Triglyceride;
aminotransferase; AST:FGF21: Fibroblast
Aspartate growth factor 21;
aminotransferase; GGT:ALT:Gamma-glutamyl
Alanine amino-
transferase; AST: Aspartate aminotransferase; GGT: Gamma-glutamyl transferase;
transferase; IL-6: Interleukin 6; IL-1β: Interleukin 1β; TNF-α: Tumor necrosis factor IL-6: Interleukin
α; GSH:
6; IL-1β: Interleukin 1β; TNF-α: Tumor necrosis factor α; GSH: Glutathione peroxidase; SOD: Su-
peroxide dismutase. Red arrow: upregulated biomarkers. Blue arrow: downregulated biomarkers.
Green minus symbol: process leads to biomarkers inhibition. Pink plus symbol: process leads to
biomarkers activation. Figure made with Servier Medical Art, [Link] (accessed
on 9 September 2023).

Fibroblast growth factor-21 (FGF21) is a molecule present in many different organs,

including the liver, and is induced by the transcription factor peroxisome proliferator-
activated receptor α (PPARα) in a state of metabolic imbalance, such as fasting [79]. FGF21
has been detected as a potential biomarker of liver steatosis as its reduction is linked to
J. Clin. Med. 2023, 12, 6561 7 of 15

the decrease in hepatic fat and enhancement of mitochondrial function [56]. Results of
the 8-week RCT by Latva-Rasku et al. confirmed the reduction in liver fat and FGF21 in
T2DM patients on dapagliflozin treatment [56]. Interestingly, a randomized controlled trial
(RCT) reported that dapagliflozin monotherapy in T2DM patients was associated with
a significant reduction in FGF21 levels, while no significant changes in placebo or dual
therapy with dapagliflozin and omega-3 carboxylic acids groups were observed [57].
Hepatic de novo lipogenesis is controlled by glucose and insulin levels by sterol
response element binding protein 1-c (SREBP1-c) and carbohydrate-responsive element-
binding protein (ChREBP) proteins [80]. Sterol response element binding protein-1a, -1c,
and -2 (SREBP-1a, -1c, and -2) are a family of integral proteins of endoplasmic reticulum
which have a central role in controlling lipid metabolism [14]. SREBP1-c, a transcription
factor regulated by insulin, is involved in de novo lipogenesis, and its overexpression in
steatosis is linked to the deposition of triglycerides in the liver [14]. It was suggested that
the sensitivity of the liver to insulin could be improved by damping lipogenesis by SREBP1
downregulation [81]. Several studies on animals have reported decreased expression
of SREBP1 when SGLT2 inhibitor was administered [5]. Mice on a diet with high fat
and empagliflozin for five weeks demonstrated lower levels of cholesterol, triglycerides,
and SREBP-1c in comparison with the control group [82]. Empagliflozin reduced the
expression of SREBP1 in another preclinical study in a rat model with no obesity or
hyperglycemia, which suggested possible benefits in clinical practice in pre-diabetes and
pre-obesity states [81].
Another participant in de novo lipogenesis is glucokinase regulatory protein (GCKR),
and the substitution of proline to leucine P446L in its encoding gene is associated with
NAFLD progression [7,83,84]. GCKRP variant P446L causes the upregulated activity of
GCKR and, consequently, elevated glycolytic flux and carbohydrate-responsive element-
binding protein (ChREBP) activity, which is a very powerful activator of de novo lipogenesis
(Figure 1) [84]. ChREBP is a transcription factor in hepatocytes, which is also triggered
by carbohydrate feeding [85]. The inhibition of ChREBP in mice with obesity and insulin
resistance led to hepatic steatosis improvement, implicating ChREBP as a contributor to
NAFLD development [14]. Interestingly, another study showed that the upregulation
of ChREBP triggers lipogenesis but without affecting glucose metabolism and insulin
resistance [86].
ChREBP controls microsomal triglyceride transfer protein (MTTP), which is located
in the lumen of the endoplasmic reticulum of hepatic cells and is a crucial factor for the
formation of lipoproteins with apolipoprotein-B and its delivery to different organs [80,87].
Because a lack of MTTP in animals and humans has been related to hepatic fat accumulation
and hypolipidemia, it is thought that MTTP can contribute to NAFLD development [80,88].
In mice models with NASH, obesity, and diabetes type 2, Honda et al. reported that
administration of ipragliflozin improved insulin resistance, triggering increased expression
of MTTP and resulting in decreased lipotoxicity [89].
Fatty acid synthase (FAS), the main enzyme of de novo lipogenesis, is controlled by
SREBP1 and is upregulated in steatosis (Figure 1) [4,5]. Furthermore, a correlation was
found between FAS expression and the severity of liver damage. Therefore, this enzyme
could be an important biomarker for the detection of liver steatosis [4]. When canagliflozin
was administered in mice, Kawarasaki et al. demonstrated reduced FAS expression relative
to fat mass, resulting in the absence of weight increase [90]. In a 24-week double-blind,
placebo-controlled clinical trial, administration of canagliflozin to patients with poorly
regulated T2DM demonstrated reduced accumulation of triglycerides within the liver [91].
Along with NAFLD development, apoptosis of hepatocytes results in the formation of
many different substrates, including keratins (K-19) and cytokeratins 18 (CK-18), which
can be detected in the blood of patients and are therefore proposed as potential biomarkers
of NAFLD progression [4,92]. Antigen CK18-M65 measures intact and cleaved CK-18, and
antigen CK18-M30 measures cleaved CK-18. The level of CK18-M30 was found to be much
higher in patients with severe steatosis, so it was suggested that CK18-M30 could help in
J. Clin. Med. 2023, 12, 6561 8 of 15

detecting patients with advanced hepatic steatosis [4]. In a double-blind, placebo-controlled

clinical trial, patients with diabetes type 2 and NAFLD receiving only dapagliflozin had
decreased both CK18-M65 and CK18-M30, which suggests a protective effect of SGLT2
inhibitors on hepatocytes in T2DM patients (Table 1) [57].
Adiponectin is an adipokine molecule predominantly produced by white adipose
tissue, and its accumulation declines as fat mass increases. This bioactive protein is found
to reduce liver steatosis and fibrosis, inflammation, and insulin resistance, and it is involved
in NAFLD progression [93,94]. Low levels of adiponectin are found to be associated with a
higher risk of T2DM, increased triglycerides, and BMI [95,96]. Some studies proposed that
SGLT2 inhibitors could elevate adiponectin levels [97,98]. This was confirmed by an RCT,
which reported increased levels of adiponectin and decreased liver fat in T2DM patients
(n = 42) after 24 weeks of empagliflozin therapy [61].

Other Potential Biomarkers of Steatosis

A common contributor of liver steatosis worsening is the modified patatin-like
phospholipase-domain containing 3 (PNPLA3) protein caused by methionine replace-
ment at residue 148 by isoleucine [99]. Elevated levels of this mutation have been observed
in those of Hispanic ethnicity, which might be the reason for a higher prevalence of NAFLD
among this population [28]. The German Diabetes Study registered as a clinical trial with
917 patients demonstrated that patients with severe insulin resistance diabetes are more
likely to be carriers of the PNPLA3 gene mutation and suggested that carriers of this
mutation have lipotoxic environments that could promote insulin resistance and NAFLD
development [100]. Mutated PNPLA3 protein has decreased hydrolase enzymatic function,
resulting in anomalous lipid remodeling and excessive lipid liver deposition [101,102].
This mechanism was confirmed in a study by Lindén et al. on mice, which showed that
decreased activity of the PNPLA3 variant leads to reduced liver fat [101,102].
The substitution of glutamate for lysine in the Human transmembrane 6 superfamily 2
(TM6SF2) mutant E167K gene is associated with incorrect folding and decomposition of the
protein TM6SF2 located in the endoplasmic reticulum and endoplasmic reticulum–Golgi
Apparatus (intermediate compartment) of liver, renal, and small intestine cells [28,103].
Protein TM6SF2 is an important player in lipid metabolism, and therefore, this mutation
contributes to metabolic disease development and is found to be associated with hepatic
steatosis progression [103]. A study on the finish population by Kim et al. demonstrated
that TM6SF2 E167K reduced serum levels of VLDL/LDL and triglycerides and therefore car-
diovascular risk but increased risk of hepatic steatosis and T2DM [104]. In 2020, Borén et al.
published research on humans, which confirmed that TM6SF2 E167K fails to export VLDL
and triglycerides from liver cells, which causes fat accumulation [105].
The systemic immune-inflammation index (SII), a newly introduced biomarker that
indicates local immune reaction and overall systemic immune inflammation, is found to be
associated with insulin resistance and could be useful in detecting NAFLD progression in
diabetes patients. A study by Xie et al. in 2022 analyzed the association between SII and
CAP and LSM in which it was found that SII value is significantly correlated with CAP, i.e.,
liver steatosis and not with LSM value, i.e., liver fibrosis [106]. This could be explained by
the fact that one-third of patients had severe fibrosis without the presence of NASH [106].
Another cross-sectional study also found a significant correlation between SII and hepatic
steatosis by measuring HIS (Hepatic steatosis index), which implies that SII could be an
important biomarker for assessing hepatic steatosis [107].

2.3. Potential Biomarkers of Fibrosis

The level of hepatic fibrosis is the most critical prognostic factor for NAFLD mortality;
therefore, fibrosis biomarkers were developed [2]. Of note, McGlinchey et al. investigated
molecules, which are specific for each stage of NAFLD and found that the most important
breaking point in the course of NAFLD progression is the transition from the F2 to F3
fibrosis stage because of the largest number of modified metabolites (n = 73) present [108]. A
J. Clin. Med. 2023, 12, 6561 9 of 15

commonly used fibrosis biomarker is the Fibrosis-4 (FIB-4) score, which is calculated based
on AST, ALT, platelet count values, and age. FIB-4 >2.67 rules in, and FIB-4 <1.30 rules
out advanced fibrosis [23,70]. Long-term outcomes were assessed in a study by Arai et al.,
including 109 patients treated with SGLT2 inhibitors for three years, demonstrating reduced
FIB-4 index, especially in patients with moderate and high risk of progressed fibrosis [26].
A superior impact on liver histology was recently proven in the RCT by Takeshita et al. due
to a significantly decreased FIB-4 score in the tofogliflozin study group [22].
A more complex biomarker for the detection of advanced fibrosis is the NAFLD fibrosis
score (NFS) resulting from albumin, age, BMI, platelet count, hyperglycemia/diabetes,
and AST/ALT ratio values [70,109]. A retrospective study by Tada et al. demonstrated
that both NFS and FIB-4 scores were associated with extrahepatic complications such as
cardiovascular and cerebrovascular diseases, suggesting that in patients with NAFLD and
T2DM, the risk of other complications and mortalities could be reduced by decreasing liver
fibrosis [26,109].
Epigenetics are of great importance to NAFLD development, progression, and clinical
pattern. A recent study regarding cardioprotective mechanisms of SGLT2 inhibitors found
that empagliflozin prevented DNA methylation induced by high glucose. Another study
associated histone post-translational modifications with SGLT2 inhibitors in diabetic kidney
disease [110,111]. However, a scarcity of current literature regarding the effects of SGLT2
inhibitors on epigenetic mechanisms shows the need for further research regarding the
effects of SGLT2 inhibitors on the epigenetics of NAFLD.

3. Future Directions
In general, the progression of NAFLD is usually asymptomatic, and there are no
specific biomarkers available in clinical practice for NAFLD diagnosis [9]. The detection of
NAFLD at an early stage is crucial for favorable disease outcomes by prevention of fibrosis.
However, early detection of a highly prevalent disease requires biomarkers that accurately
correlate with liver steatosis and fibrosis and are suitable for routine patient screening. Only
with such tools can the evaluation of the efficacy of SGLT2 inhibitors and other novel agents
be feasible. Recently, other target molecules for therapy have been described, including
the modified patatin-like phospholipase-domain containing 3 (PNPLA3) protein, human
transmembrane 6 superfamily 2 (TM6SF2), and systemic immune-inflammation index (SII).
Currently available RCTs have shown SGLT2 inhibitors’ superior efficiency in the
reduction in biomarkers such as AST, ALT, FIB-4, liver proton density fat fraction (PDFF), as
well as reduced visceral and subcutaneous fat areas, compared to other antihyperglycemic
agents (metformin, pioglitazone, sitagliptin, and glucagon-like peptide-1 receptor agonist,
and glimepiride). No superior effect, however, was determined by measuring HbA1c,
fasting plasma glucose (FPG), and the homeostasis model assessment of insulin resistance
(HOMA-IR) [112].

4. Conclusions
Evidence to date suggests that SGLT2 inhibitors play a promising role in the prevention
of NAFLD. However, whether the protective role in liver tissue is mediated by metabolic,
direct effects of the drugs themselves, or a combination of these effects remains unclear.
SGLT2 inhibitors lower blood glucose, body weight, and blood pressure on one hand and
have beneficial effects on oxidative stress, chronic inflammation, β-oxidation, de novo
lipogenesis, autophagy, and apoptosis of hepatocytes on the other. According to the clinical
trials conducted so far, there appear to be some differences in the effects on the liver
tissue between individual agents within the class. However, this could be a reflection
of different study designs or specific mechanism(s) of action and their complementary
effects on NAFLD. Therefore, the development of better biomarkers for the evaluation of
NAFLD is needed to understand the underlying mechanism of action of SGLT2 inhibitors
and other novel agents in the liver tissue. Although NAFL is considered a benign entity, it
can progress to NASH, and at present, there is no simple method of predicting those who
J. Clin. Med. 2023, 12, 6561 10 of 15

will progress. Because progression is silent and slow, the initiation of NASH is difficult
to detect with current technology. It is for this reason that biomarker development is so
important. Despite preclinical and clinical evidence that SGLT2 inhibitors may be helpful
in the treatment of NAFLD, evidence of their efficacy and long-term outcomes for this
indication is still lacking, and further large prospective double-blind and placebo-controlled
studies are needed to ultimately confirm this evidence.

Author Contributions: Conceptualization, M.S.; methodology, F.K., R.S. and M.S.; investigation, F.K.,
O.K., A.P. and L.K.R.; writing—original draft preparation, F.K., A.P. and O.K.; writing—review and
editing, F.K., K.B., A.P., I.B.-C., G.Y.W., L.K.R. and S.S.; visualization, F.K. and A.P.; supervision, H.R.,
K.B., R.S., G.Y.W., I.B.-C. and M.S.; project administration, H.R. and K.B.; funding acquisition, M.S.
All authors have read and agreed to the published version of the manuscript.
Funding: The article processing charge was funded by a grant from the Croatian Ministry of Science
and Education dedicated to multi-year institutional funding of scientific activity at the Josip Juraj
Strossmayer University of Osijek, Faculty of Medicine Osijek, Croatia—grant number IP14-MEFOS-
2022 (to M.S.).
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Data sharing is not applicable to this article as no new data were
created or analyzed in this study.
Conflicts of Interest: The authors declare no conflict of interest.

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