Endocrine Research

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Therapeutic Effects of SGLT2 Inhibitor Ipragliflozin

and Metformin on NASH in Type 2 Diabetic Mice

Atsuo Tahara & Toshiyuki Takasu

To cite this article: Atsuo Tahara & Toshiyuki Takasu (2020): Therapeutic Effects of SGLT2
Inhibitor Ipragliflozin and Metformin on NASH in Type 2 Diabetic Mice, Endocrine Research, DOI:
10.1080/07435800.2020.1713802

To link to this article: https://doi.org/10.1080/07435800.2020.1713802

Published online: 18 Jan 2020.

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ENDOCRINE RESEARCH
https://doi.org/10.1080/07435800.2020.1713802

Therapeutic Effects of SGLT2 Inhibitor Ipragliflozin and Metformin on NASH in

Type 2 Diabetic Mice
Atsuo Tahara and Toshiyuki Takasu
Drug Discovery Research, Astellas Pharma Inc., Ibaraki, Japan

ABSTRACT ARTICLE HISTORY

Background and aim: Sodium-glucose cotransporter (SGLT) 2 is responsible for most of the Received October 21, 2019
glucose reabsorption in the kidneys and has been proposed as a novel therapeutic target for the Revised November 30, 2019
treatment of type 2 diabetes. In recent years, nonalcoholic steatohepatitis (NASH), the pathogen- Accepted December 24, 2019
esis of which is strongly associated with insulin resistance, obesity, and type 2 diabetes, has KEYWORDS
become a considerable healthcare burden worldwide. However, there is currently no established SGLT2; NASH; type 2
pharmacotherapy for NASH. Here, we investigated the therapeutic effects of the SGLT2 selective diabetes
inhibitor ipragliflozin alone and in combination with metformin on NASH in high fat and
cholesterol diet-fed KK/Ay type 2 diabetic mice.
Results: This diabetic model had hyperglycemia, insulin resistance, and obesity, and also exhibited
steatosis, inflammation, and fibrosis in the liver, pathological features resembling those in human
NASH. Four-week repeated administration of ipragliflozin significantly improved not only hypergly-
cemia, insulin resistance, and obesity but also hyperlipidemia and NASH-associated symptoms
including hepatic steatosis and fibrosis. In addition, ipragliflozin attenuated inflammation and oxida-
tive stress in the liver. Repeated administration of metformin also significantly improved symptoms of
type 2 diabetes with NASH to a comparable degree to that by ipragliflozin. In addition, combination
treatment with ipragliflozin and metformin additively improved these symptoms.
Conclusions: These results demonstrate that the SGLT2 selective inhibitor ipragliflozin improves
not only hyperglycemia but also NASH in type 2 diabetic mice, suggesting that treatment with
ipragliflozin alone and in combination with metformin may be effective for treating type 2
diabetes with NASH.

Introduction 3% may reduce hepatic steatosis, up to 10% or more is

needed to reduce inflammation and for the regression
The prevalence of nonalcoholic fatty liver disease
of fibrosis in NASH patients.4 However, diet and
(NAFLD) is rapidly increasing worldwide and is cur-
other lifestyle modifications are often difficult to
rently the most common liver disorder in the Western
achieve and maintain, and this approach alone has
world.1 NAFLD describes a broad range of diseases
had limited influence on slowing the rising tide of the
from simple fatty liver, which refers to steatosis affect-
disease. In addition, NASH symptoms are further
ing hepatocytes, to nonalcoholic steatohepatitis
exacerbated if patients fail to maintain their restricted
(NASH), the inflammation and fibrosis that occurs
lifestyles. Given the challenges associated with lifestyle
in addition to steatosis which may result in hepatic
modification measures alone, combining them with
cirrhosis and hepatocellular carcinoma.2 Given that it
pharmacotherapy may be more effective for NASH
is closely related to metabolic diseases such as insulin
management. While several recent reports have
resistance, dyslipidemia, obesity, and type 2 diabetes,
described therapeutic interventions, such as metfor-
NASH can be considered a hepatic phenotype for
min and pioglitazone, for NASH,5–8 the level of evi-
metabolic syndrome.3 Lifestyle intervention to
dence remains low, and there is presently no
achieve weight loss through changes in diet and life-
established pharmacotherapy for NASH.
style habits and regular exercise is the first-line treat-
Recently, inhibitors of sodium-glucose cotranspor-
ment for NASH. While a modest weight loss of about
ter 2 (SGLT2), which stimulates glucose excretion into

CONTACT Atsuo Tahara atsuo.tahara@jp.astellas.com Drug Discovery Research, Astellas Pharma Inc., 21 Miyukigaoka, Tsukuba, Ibaraki 305-8585,
Japan
Color versions of one or more of the figures in the article can be found online at www.tandfonline.com/ierc.
© 2020 Taylor & Francis Group, LLC
2 A. TAHARA AND T. TAKASU
urine, have been proposed as novel drugs for treating
type 2 diabetes.9 Further, many studies have shown
that in addition to improving hyperglycemia, SGLT2
inhibitors also improve insulin resistance, hyperlipi-
demia, fatty liver, and obesity.10,11 SGLT2 inhibitors
are therefore expected to be effective for treating
hyperglycemia and metabolic syndromes including
NASH in type 2 diabetes. We previously investigated
the therapeutic effects of the SGLT2 inhibitor ipragli-
flozin on NASH using several animal models. In high
fat diet- and streptozotocin-nicotinamide-induced
type 2 diabetic mice, ipragliflozin significantly attenu-
ated diabetic symptoms including hyperglycemia and
obesity as well as those of NASH such as liver inflam-
mation and steatosis.12 In choline-deficient L-amino
acid-defined (CDAA) diet-induced NASH rats, ipra-
gliflozin had a prophylactic effect on liver fibrosis.13
However, because the former model does not exhibit
liver fibrosis and the latter model does not exhibit
obesity or insulin resistance, which are typical features
of NASH in humans, these studies were unable to
comprehensively evaluate the therapeutic effects of
SGLT2 inhibitors on NASH.
Here, we investigated the effects of ipragliflozin on
a number of diabetic symptoms and the progression
of NASH in high fat and cholesterol diet-fed type 2
diabetic mice that exhibit hyperglycemia, insulin
resistance, and obesity, as well as NASH-associated
symptoms including liver fibrosis. In addition, we
compared the effects of ipragliflozin with those of
metformin, and investigated the therapeutic effects
of the combination of ipragliflozin and metformin.
Materials and Methods
Materials and Animals
Ipragliflozin was synthesized at Astellas Pharma Inc.
(Ibaraki, Japan). Metformin was purchased from MP
Biomedicals (Solon, OH, USA). Drugs were sus-
pended or dissolved in 0.5% methylcellulose solution
and administered orally via a stomach tube.
Ipragliflozin was administered once daily at night
(19:00–20:00) and metformin was administered
twice daily, once at night and once in the morning
(8:00–9:00). Doses of ipragliflozin were expressed as
the free base form.
Male C57BL/6 (normal) and KK/Ay type 2 dia-
betic mice were purchased from Charles River
Laboratories Japan, Inc. (Tokyo, Japan) and CLEA
Japan (Kanagawa, Japan), respectively, at age 6 weeks
and fed either a normal chow diet consisting (as
a percentage of total calories [kcal]) of 11% fat, 69%
carbohydrate, and 20% protein (total caloric
energy = 3.7 kcal/g; D12337; Research Diets, Inc.,
New Brunswick, NJ, USA) or a high fat and choles-
terol (Paigen) diet consisting of 35% fat, 45% carbo-
hydrate, 20% protein, 12.5% cholesterol and 5%
sodium cholic acid (total caloric energy = 4.1 kcal/
g; D12336; Research Diets, Inc.). Diabetic mice with
NASH were grouped such that mean blood glucose
levels were uniform among groups. All animals were
housed under conventional conditions with con-
trolled temperature, humidity, and light (12-h light-
dark cycle) and were provided food and water ad
libitum. Animals were handled and cared for in
accordance with the Guide for the Care and Use of
Laboratory Animals, and all procedures were
approved by the Animal Ethical Committee of
Astellas Pharma Inc.
Repeated Administration Study
Vehicle, ipragliflozin (0.1–3 mg/kg), metformin
(30–300 mg/kg), or ipragliflozin (1 mg/kg) + met-
formin (100 mg/kg) was orally administered to dia-
betic mice with NASH for 4 weeks, and body weight
was measured weekly. Daily food consumption was
calculated based on the weekly difference between
the food remaining and food supplied. After drug
administration at night on Day 14, blood samples for
the evaluation of blood glucose and plasma insulin
levels were taken from the tail vein at each sampling
point for 24 h.
An oral glucose tolerance test (OGTT) was per-
formed at Week 3. After drug administration at
night on Day 22, mice were fasted for 10 h. The
next morning, blood was sampled from the tail
vein for the evaluation of fasting blood glucose
and plasma insulin levels. A glucose solution
(2 g/kg) was orally administered, and blood sam-
pling was conducted for 2 h. After drug adminis-
tration at night on Day 26, mice were transferred
to metabolic cages and spontaneously voided urine
was collected for 24 h. Two to four hours after the
final drug administration in the morning (Day 29),
blood samples were collected under nonfasting

conditions, and liver and epididymal adipose tis-
sue were isolated under isoflurane anesthesia.
Biochemical Measurements
Blood and urine glucose concentrations were mea-
sured using the Glucose CII test reagent (Wako Pure
Chemical Industries, Ltd., Osaka, Japan). Hemoglobin
A1c (HbA1c) levels were measured using a DCA2000
System (Bayer Medical, Tokyo, Japan). Plasma insulin
levels were measured using an enzyme-linked immu-
nosorbent assay (ELISA) kit (Morinaga Institute of
Biological Science, Inc., Kanagawa, Japan). Plasma
lipid concentrations and hepatic lipid contents were
measured in accordance with a previously reported
method.14 Levels of aminotransferases – alanine
amino transferase (ALT) and aspartate amino trans-
ferase (AST) – were measured using the Transaminase
CII test reagent (Wako Pure Chemical Industries,
Ltd.). Concentrations of plasma cytokines, interleukin
6 (IL-6), tumor necrosis factor α (TNF-α), and mono-
cyte chemotactic protein-1 (MCP-1), and c-reactive
protein (CRP) were measured using commercial
ELISA kits (R&D Systems Inc., Minneapolis, MN,
USA). Levels of oxidative stress biomarkers (thiobar-
bituric acid reactive substances [TBARS] and protein
carbonyl) were measured in accordance with
a previously reported method (Nakhaee et al.,15)
using commercial assay kits (Cayman Chemical
Company, Ann Arbor, MI, USA).
Histopathology
Preparation of specimens and histopathological exam-
ination were performed at CMIC Bioresearch Center
Co., Ltd. (Yamanashi, Japan). Sagittal slices of liver
fixed in 10% neutral buffered formalin were
embedded in paraffin and cut into 2-μm-thick sec-
tions for morphological study. The sections were
stained with hematoxylin and eosin (H&E) and sirius
red. All tissue samples were evaluated by an indepen-
dent investigator blinded to group information.
Microvesicular steatosis and hepatocellular hypertro-
phy were evaluated according to the percentage of area
affected: 0, <5%; 1, 5–33%; 2, 34–66%; 3, >66%.
Inflammation was evaluated by counting the number
of inflammatory foci per field: 0, normal (<0.5 foci); 1,
slight (0.5–1 foci); 2, moderate (1–2 foci); 3: severe (>2
foci). Hepatic fibrosis was evaluated using
ENDOCRINE RESEARCH 3
a semiquantitative scoring system: 0, normal; 1, slight;
2, moderate; 3, severe.
Statistical Analysis
Results are expressed as the mean ± standard error of
the mean (SEM) or individual values and median. The
area under the curve (AUC) was calculated from
blood glucose and plasma insulin concentrations
measured over time. The Matsuda index was calcu-
lated using the following formula: 10,000/square root
of [(fasting blood glucose × fasting plasma insulin) ×
(mean blood glucose × mean plasma insulin during
OGTT)], and disposition index was calculated using
the following formula: (plasma insulin AUC/blood
glucose AUC during OGTT) × Matsuda index.16
Differences between two groups were determined
using the Student’s t-test, while those among multiple
groups were assessed using Dunnett’s multiple com-
parisons test. For comparison of histopathological
scores, Mann-Whitney U test was used to analyze
differences between two groups, while Dunn’s multi-
ple comparisons test was used for comparisons
among multiple groups. P < .05 was considered sig-
nificant. Statistical and data analyzes were conducted
using GraphPad Prism 5 (GraphPad Software, La
Jolla, CA, USA).
Results
Compared to normal mice, high fat and choles-
terol diet-fed diabetic mice exhibited characteris-
tics of type 2 diabetes such as hyperglycemia,
hyperinsulinemia, glucose intolerance, hyperpha-
gia, obesity (increased body and epididymal fat
weights), glycosuria, polyuria, and dyslipidemia
(elevated plasma levels of triglycerides, non-
esterified fatty acids [NEFAs], and cholesterol)
(Table 1, Figures 1 and 2).
At Week 2 of repeated administration in the 24-h
blood collection experiment, once-daily administra-
tion of ipragliflozin (0.1–3 mg/kg) or twice-daily
administration of metformin (30–300 mg/kg)
decreased blood glucose and plasma insulin levels
in a dose-dependent manner (Figure 1). These
effects of ipragliflozin and metformin were signifi-
cant at doses of 0.3 mg/kg or higher and 100 mg/kg
or higher, respectively. Decreases in blood glucose
and plasma insulin AUCs for 24 h were similar
 4
A. TAHARA AND T. TAKASU

Table 1. Effects of single or combination treatment with ipragliflozin and metformin in type 2 diabetic mice with NASH.
Ipragliflozin +
Ipragliflozin Metformin Metformin
1 mg/kg +
Parameter Normal Vehicle 0.1 mg/kg 0.3 mg/kg 1 mg/kg 3 mg/kg 30 mg/kg 100 mg/kg 300 mg/kg 100 mg/kg
Body weight (g) 25.8 ± 0.6 47.4 ± 0.6* 47.0 ± 0.5 45.1 ± 0.6 43.0 ± 0.7# 42.1 ± 0.9# 47.1 ± 0.9 45.9 ± 0.5 44.6 ± 0.6# 42.3 ± 0.4#$
Food intake (g/day) 2.94 ± 0.07 5.04 ± 0.11* 5.13 ± 0.16 5.01 ± 0.13 5.17 ± 0.19 4.99 ± 0.22 4.91 ± 0.16 4.67 ± 0.10 4.36 ± 0.14# 4.82 ± 0.13
Epididymal adipose tissue weight (g) 0.19 ± 0.01 1.10 ± 0.05* 1.08 ± 0.07 0.91 ± 0.03# 0.82 ± 0.04# 0.68 ± 0.04# 1.02 ± 0.04 0.92 ± 0.05 0.78 ± 0.08# 0.62 ± 0.05#$
HbA1c (%) 2.95 ± 0.07 8.88 ± 0.28* 8.45 ± 0.21 7.27 ± 0.17# 6.15 ± 0.22# 5.28 ± 0.14# 8.50 ± 0.24 7.20 ± 0.31# 6.07 ± 0.36# 4.70 ± 0.21#$
Blood glucose (mg/dL) 152 ± 3 405 ± 26* 344 ± 33 288 ± 16# 231 ± 10# 187 ± 11# 356 ± 19 272 ± 28# 238 ± 11# 166 ± 18#$
Plasma insulin (ng/mL) 1.7 ± 0.1 45.2 ± 4.7* 39.0 ± 4.1 32.4 ± 2.5 24.2 ± 4.6# 20.6 ± 2.0# 39.1 ± 3.1 32.7 ± 2.4# 26.4 ± 2.0# 20.0 ± 2.8#$
Plasma triglycerides (mg/dL) 106 ± 7 346 ± 24* 301 ± 26 244 ± 28# 193 ± 11# 161 ± 12# 324 ± 16 238 ± 17# 171 ± 19# 129 ± 12#$
Plasma NEFAs (mEq/L) 0.75 ± 0.09 1.56 ± 0.14* 1.52 ± 0.15 1.21 ± 0.11 1.04 ± 0.14# 0.93 ± 0.09# 1.53 ± 0.13 1.27 ± 0.10 1.07 ± 0.14# 0.86 ± 0.07#$
Plasma cholesterol (mg/dL) 90 ± 4 198 ± 10* 192 ± 16 162 ± 9 135 ± 13# 106 ± 5# 191 ± 11 179 ± 16 148 ± 16# 91 ± 3#$
Urine volume (mL/day) 1.2 ± 0.2 4.8 ± 0.4* 4.8 ± 0.7 5.4 ± 0.4 6.2 ± 0.4 7.2 ± 0.8# 4.5 ± 0.5 3.4 ± 0.5 2.6 ± 0.3# 6.3 ± 0.6$
Urinary glucose excretion (mg/day) 1.3 ± 0.2 437 ± 49* 534 ± 81 751 ± 59# 937 ± 85# 1120 ± 120# 381 ± 30 206 ± 51# 45 ± 13# 693 ± 74#$
NEFAs: non-esterified fatty acids. Ipragliflozin and/or metformin was orally administered to diabetic mice with NASH for 4 weeks. Values are mean ± SEM for six animals per group. *P < 0.05 vs. normal
group, #P < 0.05 vs. vehicle group, $P < 0.05 vs. metformin (100 mg/kg) group.
 ENDOCRINE RESEARCH 5

a b

Figure 1. Effects of single or combination treatment with ipragliflozin and metformin on blood glucose and plasma insulin levels in
type 2 diabetic mice with NASH. Time course of changes in (a) blood glucose and (b) plasma insulin levels, and area under the blood
glucose and plasma insulin concentration-time curve (AUC) for 24 h. Blood glucose and plasma insulin levels were measured at Week
2 of repeated administration. Values are mean ± SEM for six animals per group. *P < .05 vs. normal group, #P < .05 vs. vehicle group,
$
P < .05 vs. metformin (100 mg/kg) group.

between the two drugs. Combination treatment with
ipragliflozin (1 mg/kg) and metformin (100 mg/kg)
significantly and additively reduced blood glucose
and plasma insulin levels.
In the OGTT performed at Week 3, ipragliflozin
dose-dependently improved glucose tolerance and
insulin resistance and secretion (based on the
Matsuda index and disposition index), and these
effects were significant at doses of 1 mg/kg or higher
(Figure 2). Metformin also dose-dependently and
significantly improved these parameters at 300 mg/
kg, although the effects were slightly weaker than
6 A. TAHARA AND T. TAKASU

a b

c d

Figure 2. Effects of single or combination treatment with ipragliflozin and metformin on glucose tolerance and insulin resistance in
type 2 diabetic mice with NASH. Time course of changes in (a) blood glucose and (b) plasma insulin levels, and area under the blood
glucose and plasma insulin concentration-time curve (AUC) during the oral glucose tolerance test (OGTT). The (c) Matsuda index and
(d) disposition index were used as measures of insulin resistance and secretion, respectively. OGTT was performed at Week 3 of
repeated administration. Values are mean ± SEM for six animals per group. *P < .05 vs. normal group, #P < .05 vs. vehicle group,
$
P < .05 vs. metformin (100 mg/kg) group.

those of ipragliflozin. Combination treatment with
ipragliflozin and metformin significantly and addi-
tively improved glucose tolerance and insulin
resistance.
After the 4-week repeated administration, ipragli-
flozin had dose-dependently and significantly
increased urinary glucose excretion and the associated
urine volume, and reduced nonfasting blood glucose,
HbA1c, and plasma insulin levels (Table 1). Body and
epididymal adipose tissue weights were significantly
decreased, without marked changes to food consump-
tion throughout the study period. In addition, plasma
lipid (triglycerides, NEFAs, and cholesterol) levels
were significantly decreased. Metformin also dose-
dependently and significantly reduced levels of non-
fasting blood glucose, HbA1c, plasma insulin and lipid
parameters. Unlike ipragliflozin, metformin
decreased urinary glucose excretion via attenuation
of hyperglycemia. In addition, metformin signifi-
cantly decreased body and epididymal adipose tissue
weights and food intake. Combination treatment with
ipragliflozin and metformin significantly increased
urinary glucose excretion and the associated urine
volume, and additively reduced levels of nonfasting
blood glucose, HbA1c, and plasma insulin, plasma
lipid parameters, and body and epididymal adipose
tissue weights without marked effects on food
consumption.
Ipragliflozin dose-dependently and significantly
decreased liver weight, hepatic lipid contents (tri-
glycerides and cholesterol), and plasma levels of
ALT/AST (Figure 3). Metformin also significantly
decreased these steatohepatitis parameters, and
combination treatment with ipragliflozin and met-
formin significantly and additively decreased these
parameters. Type 2 diabetic mice with NASH
exhibited marked hepatocellular hypertrophy,
microvesicular steatosis with focal lymphocytic
infiltration, intense lobular inflammation, and
mild fibrosis (Figure 4 and 5). Ipragliflozin dose-
dependently and significantly improved these signs
of liver injury including fibrosis. Metformin also
significantly attenuated these signs of liver injury,
and combination treatment with ipragliflozin and
metformin produced significant and additive
improvements.
Ipragliflozin dose-dependently and significantly
improved plasma and liver levels of proinflamma-
tory cytokines (IL-6, MCP-1, and TNF-α) and the
ENDOCRINE RESEARCH 7
inflammatory parameter CRP (Figure 6). Metformin
also significantly attenuated, or showed a trend
toward attenuating these inflammatory parameters,
and combination treatment with ipragliflozin and
metformin significantly and additively attenuated
these parameters. Ipragliflozin dose-dependently
and significantly decreased plasma and hepatic levels
of TBARS and protein carbonyl (Figure 7).
Metformin also significantly decreased these oxida-
tive stress markers, and combination treatment with
ipragliflozin and metformin significantly and addi-
tively decreased these markers.
Discussion
With the increasing prevalence of type 2 diabetes,
obesity, and metabolic syndromes due to the excessive
consumption of high fat and carbohydrate diets and
lack of exercise throughout the world, NASH is
a growing health concern with high unmet therapeu-
tic needs. However, there are currently no drugs
approved to treat patients with NASH because ther-
apeutic advances have been slow. To facilitate the
development of novel diagnostic and therapeutic
interventions for NASH, it is crucial to perform phar-
macological experiments using animal models with
clinical pathophysiology that closely resembles that of
NASH in humans. Animal models of NASH can be
divided into two main categories: those induced by
genetic mutation and those with an acquired pheno-
type produced by dietary and/or pharmacological
manipulation. Genetic leptin-deficient (ob/ob), lep-
tin-resistant (db/db), or Agouti mutation (KK/Ay)
models, and methionine/choline-deficient (MCD)
diet, CDAA diet-, high-fat diet-, or high fat and cho-
lesterol (Paigen) diet-fed models are used in the
majority of published research on NASH.17–19 We
have investigated the therapeutic effects of the
SGLT2 inhibitor ipragliflozin on NASH using several
of these animal models, namely CDAA diet-fed rats,
and high-fat diet-fed and streptozotocin-
nicotinamide-induced type 2 diabetic mice.12,13
NASH is considered a hepatic manifestation of meta-
bolic syndrome because it is strongly associated with
insulin resistance, and among the histologic features
of NASH, fibrosis is the most strongly correlated with
end-stage liver disease and mortality.20 Although the
animal models we have examined exhibit some patho-
physiologic abnormalities similar to those observed in
8 A. TAHARA AND T. TAKASU

a b

c d

Figure 3. Effects of single or combination treatment with ipragliflozin and metformin on NASH-related parameters, (a) liver weight,
hepatic contents of (b) triglycerides and (c) cholesterol, and plasma levels of (d) ALT and (e) AST, in type 2 diabetic mice with NASH.
Ipragliflozin and/or metformin was orally administered to diabetic mice with NASH for 4 weeks. Values are mean ± SEM for six
animals per group. *P < .05 vs. normal group, #P < .05 vs. vehicle group, $P < .05 vs. metformin (100 mg/kg) group.

human NASH, such as hepatic steatosis, these models
lack insulin resistance or liver histological changes
such as fibrosis, which are typical pathophysiological
features of human NASH. This places restrictions on
the translation of results obtained from these noncli-
nical models to clinical settings and subsequently the
development of therapeutic strategies for the disease.
Therefore, the most appropriate animal models of
NASH should display both metabolic abnormalities
such as obesity and insulin resistance and liver histol-
ogy including steatosis and fibrosis. Studies have
reported that dietary manipulations, such as feeding
animals with excessive fat and/or cholesterol, can
exacerbate steatosis to fibrosing steatohepatitis in
type 2 diabetic mice.21,22 Based on these reports, we
created a new NASH animal model which exhibited
both type 2 diabetic symptoms – insulin resistance
and obesity – as well as fibrosing steatohepatitis by
combining genetic type 2 diabetic mice with dietary
manipulation using a high fat and cholesterol diet.
This mouse model exhibited not only steatosis but
also inflammation and fibrosis in the liver, as well as
symptoms of type 2 diabetes such as hyperglycemia,
insulin resistance, and obesity. Therefore, this type 2
diabetes with NASH mouse model showed similar
biochemical and pathological characteristics to that
of human NASH. We used these type 2 diabetic mice
with NASH to investigate the therapeutic effects of
ipragliflozin and metformin. Once-daily administra-
tion of ipragliflozin produced long-lasting decreases
in blood glucose and plasma insulin levels. Repeated
administration of ipragliflozin reduced HbA1c levels
by increasing urinary glucose excretion. In addition,
ipragliflozin improved glucose tolerance, hyperinsu-
linemia, and insulin resistance. These improvements
by ipragliflozin are similar to those shown previously
 ENDOCRINE RESEARCH 9

Figure 4. Representative light micrographs of liver tissue obtained from (a) normal, (b) vehicle-treated, (c) ipragliflozin (1 mg/kg)-
treated, (d) metformin (100 mg/kg)-treated, and (E) ipragliflozin (1 mg/kg) + metformin (100 mg/kg)-treated type 2 diabetic mice
with NASH. Ipragliflozin and/or metformin was orally administered to diabetic mice with NASH for 4 weeks. Sections were stained
with hematoxylin and eosin (left) and sirius red (right). Magnification: x100.
10 A. TAHARA AND T. TAKASU

a b

c d

Figure 5. Effects of single or combination treatment with ipragliflozin and metformin on hepatic histopathological scores for (a)
microvesicular steatosis, (b) hepatocellular hypertrophy, (c) inflammation, and (d) fibrosis in type 2 diabetic mice with NASH.
Ipragliflozin and/or metformin was orally administered to diabetic mice with NASH for 4 weeks. Values are individual values and
median for six animals per group. *P < .05 vs. normal group, #P < .05 vs. vehicle group, $P < .05 vs. metformin (100 mg/kg) group.

in type 2 diabetes models,12 demonstrating that ipra-
gliflozin is also effective for treating diabetes symp-
toms in this type 2 diabetes with NASH model.
Repeated administration of ipragliflozin
improved hyperlipidemia and decreased epididymal
adipose tissue and body weights without affect food
intake. Administration of ipragliflozin (3 mg/kg)
increased urinary excretion of glucose by 680 mg
per day, which is equivalent to a loss of 2.3 kcal
per day or 64 kcal in four weeks. This calorie loss is
equivalent of 9 g of fat, which is sufficient to induce
the weight loss (5 g) observed in this study.
A previous study reported that ipragliflozin reduced
body fat mass by increasing lipolysis and fatty acid
oxidation in high-fat diet-induced obese rats.23
These findings suggest that ipragliflozin has thera-
peutic potential for improving lipid abnormalities
and obesity in type 2 diabetic mice with NASH.
In the present study, ipragliflozin significantly
reduced ALT and AST levels and ameliorated
hepatic steatosis as well as inflammation and fibro-
sis in type 2 diabetic mice with NASH. The pre-
valence of NASH is high in conditions associated
with insulin resistance and metabolic abnormal-
ities such as type 2 diabetes, obesity, and dyslipi-
demia. There is an increasing body of evidence
suggesting that the relationship between NASH
and insulin resistance is bidirectional. Hepatic
steatosis is significantly increased in subjects with
insulin resistance due to increased free fatty acid
flux from adipose tissue and uptake by the liver,
impaired fatty acid oxidation, and increased de
novo hepatic lipogenesis. This hepatic steatosis in
turn contributes to impaired glucose metabolism
and insulin resistance, creating a vicious cycle.24
Our results suggest that ipragliflozin improves
insulin resistance and other metabolic abnormal-
ities such as hyperglycemia and hyperlipidemia by
increasing urinary glucose excretion. In addition,
ipragliflozin induces a negative energy balance and
improves obesity. Most published studies have
shown that improving obesity using a calorie-
restricted diet and physical exercise leads to
a decrease in the incidence of insulin resistance
and metabolic syndrome and resolution of hepatic
steatosis, suggesting that this strategy should form
part of any NASH treatment regimen.25 This anti-
obesity effect of ipragliflozin may also contribute
to the improvement of NASH. Furthermore, stu-
dies have reported that SGLT2 inhibitors increase
expression of lipolytic genes, such as carnitine
palmitoyltransferase (CPT) 1α, and decrease the
expression of lipogenic genes, such as stearoyl-
CoA desaturase (SCD)-1, fatty acid synthetase
 ENDOCRINE RESEARCH 11

Figure 6. Effects of single or combination treatment with ipragliflozin and metformin on plasma (left) and hepatic (right) levels of (a)
IL-6, (b) MCP-1, (c) TNF-α, and (d) CRP in type 2 diabetic mice with NASH. Ipragliflozin and/or metformin was orally administered to
diabetic mice with NASH for 4 weeks. Values are mean ± SEM for six animals per group. *P < .05 vs. normal group, #P < .05 vs.
vehicle group, $P < .05 vs. metformin (100 mg/kg) group.

(FAS), sterol regulatory element-binding protein
(SREBP)-1c, and diacylgycerol acyltransferase
(DGAT), in the liver.26,27 These mechanisms may
also contribute to the attenuation of hepatic stea-
tosis and progression of NASH by SGLT2 inhibi-
tors including ipragliflozin.
A previous study proposed that the development
of NASH was a “two hit” process.20 The first hit is the
above-mentioned hepatic steatosis and the second
hit is the induction of hepatic inflammation and
fibrosis, and includes cellular stresses such as oxida-
tive stress, apoptosis, and inflammation.28 Among
these physiological changes, increased plasma levels
of proinflammatory cytokines such as TNF-α and
IL-6 are common in NASH patients.29 These inflam-
matory factors, which originate largely from adipose
tissue, worsen insulin resistance and promote liver
injury. In addition, oxidative stress, mainly caused by
mitochondrial dysfunction, is thought to play an
important role in the progression of liver damage
in NASH.30 In this study, ipragliflozin potently atte-
nuated plasma and hepatic levels of inflammatory
and oxidative stress markers. Therefore, the benefi-
cial effects of ipragliflozin are not limited to the
amelioration of hepatic steatosis, but extend to
reductions in inflammation, oxidative stress, and
12 A. TAHARA AND T. TAKASU

Figure 7. Effects of single or combination treatment with ipragliflozin and metformin on oxidative stress markers, and plasma (left)
and hepatic (right) levels of (a) TBARS and (b) protein carbonyl in type 2 diabetic mice with NASH. Ipragliflozin and/or metformin
was orally administered to diabetic mice with NASH for 4 weeks. Values are mean ± SEM for six animals per group. *P < .05 vs.
normal group, #P < .05 vs. vehicle group, $P < .05 vs. metformin (100 mg/kg) group.

fibrosis. To our knowledge, this is the first study to
demonstrate the favorable effects of an SGLT2 inhi-
bitor on insulin resistance, inflammation, oxidative
stress, hepatic steatosis, and fibrosis using a type 2
diabetes with NASH model. However, to estimate
the clinical effects of ipragliflozin on type 2 diabetes
with NASH, additional detailed examinations will be
necessary, including the use of a range of drug doses
and administration periods.
Metformin, a biguanide, remains the most widely
used first-line drug for the treatment of type 2 dia-
betes. It has been shown to improve insulin resistance
and hyperglycemia via activation of the AMP-
activated protein kinase (AMPK) pathway, and
thereby enhances peripheral glucose uptake and
reduces hepatic glucose production.31 Many studies
have evaluated the use of agents such as metformin
and pioglitazone for improving insulin resistance as
a possible treatment for NASH.32,33 To date,
a number of clinical studies have investigated the
efficacy of metformin on NASH, with several trials
indicating beneficial effects in patients with NASH.34
Marchesini et al.35 reported that metformin signifi-
cantly reduced plasma ALT levels and attenuated
hepatomegaly. In contrast, while most clinical studies
have shown that metformin improves serum liver
enzymes and insulin resistance, evidence on its effec-
tiveness in ameliorating liver histological injury is
inconsistent.7 Therefore, due to its metabolic effects
and safety profile, metformin can attenuate hepatic
steatosis and metabolic abnormalities including insu-
lin resistance, and remains a promising drug for
NASH therapy, although whether it ameliorates hepa-
tic pathological injuries including fibrosis in NASH
remains unclear.
In this study, twice-daily administration of met-
formin significantly decreased blood glucose and
plasma insulin levels. Repeated administration of
metformin not only reduced HbA1c levels but also
improved glucose tolerance, hyperinsulinemia, and
insulin resistance. The effectiveness of metformin
as an antidiabetic drug is due to its ability to lower
blood glucose and plasma insulin levels by
decreasing gluconeogenesis in the liver, stimulat-
ing glucose uptake in the muscle, and increasing
fatty acid oxidation in adipose tissue via activation
of AMPK, a master regulator of glucose and lipid
metabolism.31 A reduction in hyperinsulinemia is
related to improvements in insulin resistance.
Repeated administration of metformin improved

hyperlipidemia and decreased epididymal adipose
tissue and body weights, as well as in food intake.
Weight loss during metformin treatment has been
mainly attributed to a decrease in net caloric
intake, probably through appetite suppression.36
These data support the rationale for use of met-
formin, which helps to reduce body weight in
obese type 2 diabetic patients and contributes to
significantly reducing body fat.
In the present study, metformin significantly
reduced plasma aminotransferase levels and ame-
liorated hepatic steatosis. In addition, metformin
attenuated inflammation, oxidative stress, and
pathological injuries including fibrosis. While
these therapeutic effects of metformin on NASH
are consistent with the results of nonclinical
studies,37 they are not fully consistent with clinical
findings.7 Because insulin resistance plays a central
role in type 2 diabetes with NASH, these effects of
metformin are likely to have a favorable effect on
hepatic steatosis. In addition, a study reported that
metformin suppressed oxidative stress and
restored the antioxidant capacity of the liver,33
which may result in improved hepatic lipid meta-
bolism and improved steatosis and renal injury.
Combination treatment with ipragliflozin and
metformin significantly and additively improved
symptoms of diabetes and metabolic abnormalities
as well as NASH. Thus far, we have reported that
combination treatment with ipragliflozin and met-
formin additively enhances the improvement in glu-
cose tolerance and hyperglycemia without affecting
each drug’s unique pharmacological effects, includ-
ing the increase in urinary glucose excretion by
ipragliflozin and decrease in hepatic phosphoenol-
pyruvate carboxykinase (PEPCK) activity by
metformin.38,39 Results of this study suggest that
both drugs exhibited attenuation of inflammation,
oxidative stress, hepatic steatosis, and fibrosis in type
2 diabetes with NASH mice via the direct improve-
ment of hyperglycemia and by the secondary
improvement of other type 2 diabetic symptoms,
including insulin resistance. In addition, SGLT2
inhibitors attenuated NASH by the improvement of
glycolipid metabolism in the whole body, including
liver via the urinary excretion of excess glucose; and
metformin attenuated NASH by a direct effect on
several glycolipid metabolism pathways, including
PEPCK and adenosine monophosphate-activated
ENDOCRINE RESEARCH 13
protein kinase (AMPK) in the liver.34 The present
study demonstrated for the first time that the com-
bination of ipragliflozin and metformin additionally
prevented the development of NASH.
As mentioned above, metformin significantly
attenuated aminotransferase levels and hepatic stea-
tosis as well as inflammation and fibrosis in this type
2 diabetes with NASH model, which is inconsistent
with clinical observations. This discrepancy between
findings in rodents and humans may result from
differences in pathogenesis, genotype, and con-
founding factors intrinsic to rodents. Another expla-
nation may be the need for different effective doses
of metformin for improving hyperglycemia and
NASH. In this type 2 diabetes with NASH model,
metformin exerted significant antihyperglycemic
effects at doses of 100 mg/kg or higher. In contrast,
metformin only significantly improved insulin resis-
tance and NASH at the highest dose of 300 mg/kg.
These findings suggest that a high dose of metformin
may be necessary to improve insulin resistance and
NASH. However, Haukeland et al. (2009) reported
that high doses (2.5–3 g/day) of metformin did not
improve NASH. In addition, high doses of metfor-
min lead to side effects including lactic acidosis.
Results using a high dose of metformin in this
study will be valuable in evaluating the efficacy of
metformin for improving NASH; nevertheless,
further investigation and discussion is required to
clarify these issues and to estimate the degree of
clinical usefulness against NASH. In contrast, ipra-
gliflozin improved hyperglycemia, insulin resistance,
and NASH at the same dosage. Therefore, ipragli-
flozin may be effective for improving NASH and
diabetes in clinical settings. In addition, our findings
suggest that combinatorial administration of ipragli-
flozin and metformin may be an excellent therapeu-
tic treatment for type 2 diabetes and NASH. The
effectiveness of this treatment regimen should be
examined in clinical studies. Furthermore, the type
2 diabetes with NASH model mice created in
a relatively short experimental period in this study
will be useful in investigating the pathophysiology of
NASH and estimating the pharmacological effects of
various antidiabetic drugs. In contrast, the high fat
and cholesterol diet used in this study represents
a markedly different condition from that of human
diets, particularly with regard to cholesterol content.
Thus, these type 2 diabetes with NASH mice were
14 A. TAHARA AND T. TAKASU
created by feeding with special diets, and the influ-
ence of this diet on lipid metabolism warrants sui-
table consideration. In addition, several other NASH
models fed with an MCD, CDAA, or amylin-
containing high fat diet might also cause NASH
with fibrosis, and would accordingly be useful for
investigating the pathophysiology of NASH. Further
investigation of these means of inducing fibrosing
steatohepatitis will aid the development of additional
appropriate and valuable NASH animal models.40
In summary, the type 2 diabetes with NASH
mouse model recapitulates the various phenotypes
of NASH including hepatic steatosis and fibrosis and
their associated metabolic characteristics such as
insulin resistance and obesity. We hope that it will
serve as a relevant model for identifying pharmaco-
logic targets/agents, progression mechanisms, and
therapeutic approaches for NASH. Ipragliflozin atte-
nuated type 2 diabetic symptoms including hypergly-
cemia, obesity, and insulin resistance as well as the
pathophysiological progression of NASH. Further,
the beneficial effects of the SGLT2 inhibitor ipragli-
flozin were not limited to the amelioration of hepatic
steatosis, but extended to the reduction of inflamma-
tion and fibrosis. In addition, combination treatment
with ipragliflozin and metformin additively
improved these therapeutic effects. Therefore, ipra-
gliflozin alone or in combination with metformin
prevented development of the pathological features
of NASH, suggesting that it may be a new therapeutic
strategy for NASH associated with type 2 diabetes.
Acknowledgments
The authors thank Drs. Yuichi Tomura, Akiyoshi Shimaya,
Shoji Takakura, and Toshihide Yokoyama (Astellas Pharma
Inc.) and Drs. Hiroshi Tomiyama, Akira Tomiyama, and
Yoshihiko Haino (Kotobuki Pharmaceutical Co., Ltd.) for
their valuable comments and continuing encouragement.
Declaration of Interest
The authors declare no conflict of interest other than being
employees of Astellas Pharma Inc. that could be perceived as
prejudicing the impartiality of the research reported.
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