Received: 30 March 2020 Revised: 12 May 2020 Accepted: 28 May 2020

DOI: 10.1111/obr.13081

OBESITY COMORBIDITIES/ANIMAL MODELS

Diet-induced rodent models of obesity-related metabolic

disorders—A guide to a translational perspective

Inês Preguiça1,2,3 | André Alves1,2,3 | Sara Nunes1,2,3 |

1,2,3 1,2,3,4,5
Rosa Fernandes | Pedro Gomes | Sofia D. Viana1,2,3,6 |
Flávio Reis1,2,3

1
Institute of Pharmacology and Experimental
Therapeutics, and Coimbra Institute for Summary
Clinical and Biomedical Research (iCBR), Diet is a critical element determining human health and diseases, and unbalanced
Faculty of Medicine, University of Coimbra,
Coimbra, Portugal food habits are major risk factors for the development of obesity and related meta-
2
Center for Innovative Biomedicine and bolic disorders. Despite technological and pharmacological advances, as well as inten-
Biotechnology (CIBB), University of Coimbra,
sification of awareness campaigns, the prevalence of metabolic disorders worldwide
Coimbra, Portugal
3
Clinical Academic Center of Coimbra (CACC), is still increasing. Thus, novel therapeutic approaches with increased efficacy are
University of Coimbra, Coimbra, Portugal urgently required, which often depends on cellular and molecular investigations using
4
Department of Biomedicine, Faculty of
robust animal models. In the absence of perfect rodent models, those induced by
Medicine, University of Porto, Porto, Portugal
5
Center for Health Technology and Services
excessive consumption of fat and sugars better replicate the key aspects that are the
Research (CINTESIS), University of Porto, root causes of human metabolic diseases. However, the results obtained using these
Porto, Portugal
6
models cannot be directly compared, particularly because of the use of different
ESTESC-Coimbra Health School, Pharmacy,
Polytechnic Institute of Coimbra, Coimbra, dietary protocols, and animal species and strains, among other confounding factors.
Portugal This review article revisits diet-induced models of obesity and related metabolic
Correspondence disorders, namely, metabolic syndrome, prediabetes, diabetes and nonalcoholic fatty
Flávio Reis, PhD, Faculty of Medicine, University liver disease. A critical analysis focused on the main pathophysiological features
of Coimbra, 3000-548 Coimbra, Portugal.
Email: freis@fmed.uc.pt of rodent models, as opposed to the criteria defined for humans, is provided as a
practical guide with a translational perspective for the establishment of animal
Funding information
This work was supported by European models of obesity-related metabolic diseases.
Regional Development Fund (FEDER), through
Programa Operacional Factores de KEYWORDS
Competitividade COMPETE2020 (CENTRO-
diabetes, diet-induced rodent models, metabolic syndrome, nonalcoholic fatty liver disease,
01-0145-FEDER-000012-HealthyAging2020,
POCI-01-0145-FEDER-007440 and POCI- obesity, prediabetes
01-0145-FEDER-031712) and by National
funds via Portuguese Science and Technology
Foundation (FCT): Strategic Projects UID/
NEU/04539/2013, UID/NEU/04539/2019,
UIDB/04539/2020 and UIDP/04539/2020
(CIBB); PhD Fellowship SFRH/BD/109017/
2015; PTDC/SAU-NUT/31712/2017.

ABBREVIATIONS: ADA, American Diabetes Association; BMI, body mass index; BP, blood pressure; BW, body weight; CD, cafeteria diet; CDAA, choline-deficient L-amino acid; CHO,
carbohydrates; CVD, cardiovascular disease; DIO, diet-induced obesity; FBG, fasting blood glucose; FFA, free fatty acids; IFG, impaired fasting glucose; IGT, impaired glucose tolerance; ITT,
insulin tolerance test; HbA1c, glycated haemoglobin; HCC, hepatocellular carcinoma; HDL, high-density lipoprotein; HFD, high-fat diet; HFHSD, high-fat high-sugar diet; HSD, high-sugar diet;
HFHFD, high-fat and high-fructose diet; HOMA-IR, homeostatic model assessment of insulin resistance; MCD, methionine-choline-deficient; MetS, metabolic syndrome; NAFLD, nonalcoholic
fatty liver disease; NASH, nonalcoholic steatohepatitis; OGTT, oral glucose tolerance test; QUICKI, quantitative insulin check index of insulin sensitivity; STZ, streptozotocin; T2DM, type
2 diabetes mellitus; TGs, triglycerides; TWD, total Western diet; VLDL, very low-density lipoprotein; WAT, white adipose tissue; WC, waist circumference; WD, Western diets; WHO, World
Health Organization.

Inês Preguiça and André Alves contributed equally.

Obesity Reviews. 2020;1–29. wileyonlinelibrary.com/journal/obr © 2020 World Obesity Federation 1

2 PREGUIÇA ET AL.
1 | I N T RO D UC TI O N
1.1 | Overview of the problem and challenges
Globally, obesity has reached epidemic proportions, affecting not
only adults but also an increasing number of children and adoles-
cents.1
Furthermore, as obesity can progressively cause and/or
exacerbate a broad spectrum of chronic diseases, including metabolic
syndrome (MetS), prediabetes, type 2 diabetes mellitus (T2DM), non-
alcoholic fatty liver disease (NAFLD), cardiovascular disease (CVD)
2
and some types of cancer, it poses an increased risk for all-cause
mortality.3,4
Although lifestyle changes, such as calorie restriction and regular
physical activity/exercise, are effective in reducing obesity and associ-
5–7
ated complications, the majority of the population may not adhere
to these interventions. In addition, despite decades of extensive
research, only few molecules progressed successfully into approved
drugs for weight management in patients with obesity.8,9 Thus,
approaches for overcoming obesity-related metabolic disorders and
complications remain an open challenge.
Various animal models, mostly involving rodents, have been used
for several decades as fundamental tools for studying obesity, T2DM
10–14
and associated metabolic disorders. However, unlike human
diseases, the diagnostic criteria have not been clearly established for
animal models of human diseases, and the research community is
aware of their considerable drawbacks and limited predictability.15
The absence of rigorous, standardized and clinically relevant criteria
continues to hamper the translation of preclinical metabolic research
findings into drug discovery programs regarding obesity and related
metabolic disturbances and preclude biological understanding of
the disease.
Therefore, in this review, we have highlighted the main fea-
tures of dietary rodent models, alone or in combination with
chemical or genetic manipulations, which can be used to experi-
mentally replicate human obesity and associated complications,
specifically MetS, prediabetes, T2DM and NAFLD. We further pro-
vide an in-depth comparison of the main variables for the diagnosis
of diseases in humans and characterization of animal models. Reso-
lution of the prevailing discrepancies is expected to improve the
reliability of preclinical studies and thereby improve their transla-
tion to the clinic.
1.2 | Diet as a driving force for the continuum of
obesity-related metabolic disorders
Although genetic background plays a pivotal role in the establishment
of obesity, lifestyle features such as physical inactivity and chronic
hypercaloric dietary patterns are currently envisaged as the main
drivers behind the exponential increase in obesity-related metabolic
16–18
diseases. Several risk factors and pathophysiological pathways of
MetS, prediabetes, T2DM and NAFLD are overlapping.19–21 Apart
from few cases where the aforesaid disorders evolve without major
body weight (BW) changes, obesity strongly exacerbates common
pathophysiological traits involving insulin resistance, hyperglycaemia
and dyslipidaemia.22–24 For instance, excess circulating free fatty acids
(FFA) arising from enlarged adipose tissue may compete with glucose
as the substrate in peripheral tissues, fuelling insulin resistance. In
addition to impaired glucose uptake by these tissues, increased circu-
lating FFA may also impair pancreatic β-cell function and insulin secre-
tion.22,24,25 In fact, the transition from prediabetes to overt diabetes
relies mostly on the loss of a long period of increased hyper-
insulinaemia to compensate for insulin resistance.24,26 At the end
stage, β-cell failure and decreased insulin secretion further aggravate
abnormal hyperglycaemia.27 Similarly, a vicious cycle arising from
glucotoxicity and lipotoxicity pathways underlies MetS and NAFLD
pathogenesis. Overall, oxidative imbalance, low-grade inflammation,
plasmatic and/or hepatic dyslipidaemia, impaired incretin effect, gut
microbiota changes, endoplasmic reticulum stress and/or mitochon-
drial dysfunction are common cellular denominators of obesity-related
metabolic diseases.28–31
Similar to that in humans, chronic consumption of diets rich in fat
and sugar leads to deleterious metabolic effects in rodents, despite
differing responsiveness among species and strains. Thus, paradigms
of longstanding free access to palatable calorie-dense food are widely
used in preclinical research to model the progression of obesity and
related comorbidities.10,32 One approach for studying the develop-
ment of obesity in rodents involves the feeding of cafeteria diet (CD).
This method, created to closely emulate human dietary patterns,
allows animals to freely select from a variety of tasty processed foods
(e.g., cookies, cake and salami) mimicking the hedonic hyperphagia
frequently observed in humans with obesity.10,33 Although the cafete-
ria paradigm promotes pronounced weight gain and severe symptom-
atology of obesity-related metabolic diseases, the poor monitoring of
rodents' energy/nutrients/fibre intake in these regimens has hindered
its reliability.34
To ensure experimental reproducibility and improve translatabil-
ity, standardized laboratory animal diets formulated with purified raw
materials and accurately reflecting the macronutrient and micronutri-
ent content are being used. The most frequently used are high-fat
diet (HFD), high-sugar diet (HSD) and the respective combinations of
high-fat high-sugar diet (HFHSD), collectively termed “Western diets”
(WD).35
Typically, HFD (45% and 60% calories from fat) consistently
induces obesity, insulin resistance, hypertriglyceridaemia, liver
steatosis, hypertension, adipocyte hypertrophy and impairment of the
intestinal barrier/metabolic endotoxaemia; the observed variations
are largely due to the fat used (percentage and source: animal
vs. vegetable), the duration of the protocol and the rodent
species.36–41 HSD are the most diverse, as they can be administered
on their own or as a supplement to HFD. Owing to their palatability
and high energy density, sugar-based diets also promote positive
energy balance.10 Sucrose, glucose and fructose are the most preva-
lent sugar sources, although they differ considerably in terms of meta-
bolic disruption.42 For instance, ad libitum access to fructose in
beverages increases hepatic lipid accumulation and adiposity in mice
PREGUIÇA ET AL.
compared with a similar sucrose paradigm.43 Yet, HSD in rodents gen-
erally affects BW gain and adiposity lesser than HFD paradigms,
irrespective of their equally negative impact on glycaemic control.10
It is important to highlight that most of the available diets do not
closely recapitulate Western human dietary patterns, although they
can replicate some of the metabolic diseases associated with this
44
human dietary regimen in rodents. For instance, humans typically
consume 30% of their calories from fat, which is significantly lower
than the fat content observed in most of the commercially available
standardized rodent HFD.10 Therefore, experimental protocols with
45% calories from fat are more relevant for translational purposes.
However, important preclinical evidence has been obtained from diets
where 60% calories were obtained from fat, probably due to an accel-
erated disease phenotype and inherent economic perks.35
Another important feature relies on the negligible resemblance in
terms of micronutrients and additive content of human WD pattern
with the corresponding rodent's CD and WD regimens. To overcome
these gaps, experimental diets aimed to better reproduce the basic
features of Western nutrition have been recently developed. The total
Western diet (TWD), reflecting the median nutrient intake among the
population of the United States, displays energy density similar to cer-
tain commercial diet-induced obesity (DIO) formulations. However,
TWD was developed to achieve more diversity in terms of fat sources
and balanced micronutrient composition, as well as increase in com-
plex carbohydrate (CHO) and simple sugar content. Surprisingly,
TWD-fed mice did not show increase in energy intake, fat mass and
BW and failed to acquire an MetS phenotype.45 Hence, dietary micro-
nutrients may play a major role in the hyperphagic response, increas-
ing weight gain in mice.45 Another attempt to more realistically
replicate the human WD pattern in rodents involves the use of a new
prototypic animal WD formulated with high amounts of simple CHO,
salt and fat (42.5% kcal from fat), along with a low-fibre percentage.46
This novel dietary paradigm is additive free, nutritionally adequate and
highly reproducible, as it overcomes food heterogeneity issues, unlike
46
the CD regimens. Interestingly, this standardized WD approach
effectively induced excess body fat accumulation as well as obesity-
related features (e.g., hepatic steatosis and fasting hyperglycaemia),
which were independent of gut microbiota dysbiosis.46 Remarkably,
WD-fed rats gained significantly more weight and showed higher adi-
posity index than rats fed two traditional obesogenic regimens, the
CD and the HFD, obtaining 42.5% and 60% kcal from fat, respec-
tively.46 Considering the same micronutrient base of the aforemen-
tioned dietary paradigms, this elegant work pinpoints the importance
of diet choice when an obesogenic paradigm is to be selected.
In sum, subtle changes in rodent dietary paradigms (e.g., nutrient
sources, diet and physical form) may considerably affect the extent of
metabolic impairments. Hence, appropriate hypercaloric diets should
be carefully selected (and the corresponding control diets, as dis-
cussed later) to successfully achieve most of the desired experimental
outcomes. The following sections provide an in-depth analysis of the
most common dietary paradigms used to experimentally replicate the
phenotypic features comprising obesity, MetS, prediabetes, T2DM
and NAFLD in rodents.
3
2 | D I E T - I N DU C E D R O D E N T M O D E LS
2.1 | Obesity
Obesity is defined by the World Health Organization (WHO) as
abnormal or excessive fat accumulation that may impair health and is
considered a chronic relapsing progressive disease process by the
World Obesity Federation.47,48 The worldwide prevalence of obesity
has nearly tripled from 1975 to 2016, affecting 13% of the world's
adult population.49
In humans, anthropometric measures, particularly body mass
index (BMI), calculated as weight divided by the square of the height
(kg/m2), and waist circumference (WC), have been used to define indi-
viduals with overweight or obesity. In addition, body fat distribution,
besides the total amount of body fat, is an important risk factor for
obesity-related disorders.50 In particular, excess visceral adipose tis-
sue, that is, the accumulation of adipose tissue within the omentum
and abdomen and around abdominal organs, is more strongly associ-
ated with the development and progression of cardiometabolic dis-
eases in humans than subcutaneous adiposity.51 Unfortunately, in
clinical practice, as with the routine assessment of WC, body fat distri-
bution is rarely evaluated,52,53 irrespective of the methodologies
available,54–57 although the majority of the methods are laborious and
require expensive technology.
Unlike in humans, obesity has not been defined in experimental
animals. Obesity is usually considered as any significant increase in
the levels of a marker relative to that in control animals (Figure 1).
Several complementary markers have been used to assess obesity in
rodents, including body composition analysis, condition index (which
can be evaluated from the BMI using the Quételet's index, for exam-
ple), total BW gain, weight of adipose tissue depots and adipocyte
size.10,58 Imaging techniques for assessing adipose tissue distribution
are also available for laboratory animals.10 The susceptibility of ani-
mals to obesity development is highly heterogeneous, being mostly
dependent on strain, gender, age and diet composition. Therefore, we
reasoned that the establishment of comprehensive and definitive
guidelines for obesity assessment in experimental animals will contrib-
ute to advancements in the field. Although the goal of this review is
obviously not to determine the criteria for defining obesity in rodents,
data that demonstrate the diversity of parameters used in different
studies, as well as the wide range of observed results, might be poten-
tially useful. Above all, we intend to stimulate a more in-depth discus-
sion on this subject.
Several animal models, particularly rodents, have been used to
replicate the phenotype and pathogenesis of human obesity.10,13,14,59
The most commonly used obesity models can be subdivided into
those based on mutations or manipulations of one or a few individual
genes (e.g., ob/ob mouse or Zucker fatty rat [fa/fa]), or those resulting
from exposure to obesogenic environments.10,59 Despite being a com-
plex and multifactorial disorder, excess calorie intake and a sedentary
lifestyle are recognized as the main drivers of obesity. Therefore, DIO
models have gained increased relevance, as they mimic the process of
obesity development in humans more closely than genetic models.
4 PREGUIÇA ET AL.
The exposure of rodents to energy-dense diets enriched in fats
often results in obesity and related complications, even with differ-
ences in terms of dietary composition and duration of intervention.
The development of obesity in this model is thought to result from
various interrelated factors, although excess calorie intake is a major
contributor. Some strains of rats and mice are prone to the develop-
ment of DIO. For example, Sprague–Dawley and Wistar rats fed a diet
containing 40–60% energy as fat normally take 8–12 weeks to
develop obesity.41,60,61 Similarly, several inbred mouse strains, notably
C57BL/6J, FVB/N and DBA/2J, are also susceptible to HFD-induced
62–67
obesity. Interestingly, a small fraction of rodents are resistant to
DIO, although the underlying mechanisms are currently not
completely understood.68,69 However, critical information has been
obtained using obesogenic diets for the selection of obesity-prone
(OP) or obesity-resistant (OR) or DIO/diet-resistant (DR) rodents
using outbred and/or inbred strains,68,70,71 as will be mentioned
throughout the manuscript.
Regarding HFD-induced obesity, studies in Sprague–Dawley rats
with 4 weeks of feeding showed distinct percentage of BW gain (cal-
culated by discounting the change in BW of control animals) and adi-
72
pose tissue rise: 25% BW and 13% white adipose tissue (WAT)
versus 33% BW and 6.3% fat mass.73 Curiously, two other studies on
74
Wistar rats showed highly divergent percentage of BW gain (22%
and 50%75), without information regarding changes on adipose tissue
depots. The former study, however, was performed with young ani-
mals, which might explain the differences. For longer protocols
(10 weeks), another study showed major differences between Wistar
and Sprague–Dawley rats regarding both the percentage of BW gain
(22% vs. 14%) and percentage of fat mass increase (43% vs. 21%).60
An even longer protocol of 25 weeks of HFD on Sprague–Dawley rats
76
reported 32% BW gain, which is almost identical to that achieved
(33%) by other studies with only 4 weeks under HFD.73 Disparity
among research findings was also observed in other rodent species,
F I G U R E 1 Comparison of human
diagnosis criteria for obesity,
metabolic syndrome, prediabetes,
diabetes and nonalcoholic fatty liver
disease with markers used in
preclinical research to define rodent
models of those conditions. ALT,
alanine aminotransferase; AST,
aspartate transaminase; BMI, body
mass index; FBG, fasting blood
glucose; GTT, glucose tolerance test;
HbA1c, glycated hemoglobin; HDL,
high-density lipoprotein; HOMA-IR,
homeostatic model assessment of
insulin resistance; IFG, impaired
fasting glucose; IGT, impaired glucose
tolerance; IR, insulin resistance; ITT,
insulin tolerance test; OGTT, oral
glucose tolerance test; SBP, systolic
blood pressure; TGs, triglycerides
such as mice. A study using a protocol of HFD for 8 weeks in C57BL6
was associated with 16% increase in BW,77 whereas another study
performed using the same mouse strain but with 5 weeks of HFD
induced 37% increase in BW.78 On the contrary, AKR/J and 129S1/
SvlmJ mice under 10 and 20 weeks of HFD, respectively, showed
35% and 37% BW gain, although information regarding adipose tissue
alterations were lacking.79
Supported by strong epidemiological and experimental evidence
associating increased sugar consumption to the obesity epidemic,80,81
diets with high sucrose or high fructose content have also been used
as parts of the nutritional strategy for obesity induction in rodents.
For example, male NMRI outbred mice with free access to fructose-
sweetened water for 2.5 months exhibited increased BW and adipos-
ity compared with control animals.43 Similarly, both male and female
Sprague–Dawley rats maintained for 6–7 months with access to fruc-
tose water gained significantly more weight than control animals.82
However, compared with that observed on HFD, obesity marker
levels in male C57Bl/6 mice on HSD for 3 or 12 months are typically
less exacerbated and more variable, although with a similar negative
impact on glucose homeostasis.83,84 Several studies have also
assessed the potential effect of the interaction between excess fat
and sugar in diets on metabolic risk factors. Indeed, compared with
HFD alone, feeding male C57Bl/6 mice and Wistar rats with HFHSD
for 4 and 8 weeks, respectively, resulted in rapid onset and more
severe metabolic phenotype,85,86 including MetS, prediabetes, T2DM
and NAFLD, which will be discussed in the following sections.
Overall, these studies clearly demonstrated the diversity of out-
comes in models of obesity induced by hypercaloric diets, strongly
recommending a standardization of criteria, markers and cut-offs for
better comparison of results. This aspect should not be confused with
the existence of diverse models that does not constitute a problem
per se. On the contrary, it allows higher representation of heteroge-
neous human diseases.
PREGUIÇA ET AL.
2.2 | Metabolic syndrome
MetS, originally referred to as syndrome X, is roughly characterized
by abdominal obesity, insulin resistance, hypertension and hyper-
lipidaemia.87 MetS is not simply a disease, but a screening tool,
where a complex cluster of metabolic abnormalities collectively pre-
dict the risk of individuals to develop CVD, T2DM and NAFLD, as
87,88
well as kidney and pancreatic disorders. In fact, the International
Diabetes Federation (IDF) for Global Consensus Definition, in coop-
eration with the American Heart Association/National Heart, Lung,
and Blood Institute, specified in 2009 that MetS is achieved when
three out of the following five parameters are present89: (i) obesity,
with sex- and ethnicity/race-specific WC cut-offs; (ii) elevated levels
of triglycerides (TGs): ≥150 mg/dl; (iii) reduced high-density lipopro-
tein (HDL) cholesterol: <40 mg/dl in men and <50 mg/dl in women;
(iv) elevated blood pressure (BP): systolic BP ≥ 130 mm Hg or dia-
stolic BP ≥ 85 mm Hg; (v) elevated fasting blood glucose (FBG):
≥100 mg/dl (Figure 1).
The global prevalence of MetS is unknown; however, as it is
about three times more common than diabetes, it can be estimated to
affect approximately one quarter of the world population.90,91 The
prevalence varies considerably between distinct world regions, with a
92
variation of 29–40% across different states of the United States, for
92,93
example, and between sex. Despite the heterogeneity of epidemi-
ological studies with distinct populations, MetS prevalence appears to
consistently increase with age, and hypertension appears to be the
90,91
most prevalent underlying feature of MetS across countries.
Although some genetic differences may account for distinct preva-
lence of MetS among distinct communities/ethnicities,94,95 the
increased consumption of high-fat low-fibre meals and sedentary life-
96–98
style appear to be the key factors.
Considering these numbers, the establishment of animal models
of MetS for proper understanding of pathophysiological features and
therapeutic opportunities is of utmost importance for global public
health. Nevertheless, criteria for model validation have to be defined,
as otherwise, disparities in results that cannot be compared will be
continuously generated. In the absence of an institutional definition
(by a consensus group, for example), Cheng et al. 61 suggested certain
criteria to validate an experimental model of MetS, which included
weight gain, increased visceral fat mass, impaired FBG, hypertension,
hypertriglyceridaemia, elevated non-HDL cholesterol level and hepatic
steatosis. Simultaneously, food and water consumption should be
monitored rigorously to determine the precise caloric intake of each
experimental group, a key factor for ensuring model validation and
scientific replication of data.
When inducing animal models of MetS, a panoply of protocols
has been used either solely via diet manipulation or in combination
with streptozotocin (STZ), a cytotoxic drug that elicits clear
hyperglycaemia.99–101 Nonetheless, as previously mentioned, we have
herein focused on diet-induced models of MetS (Table 1), which are
mainly based on high fat, high sugar or a combination of both. HFDs
are commonly used for diet-induced metabolic abnormalities in both
rats and mice, inducing a wide range of fat content (30–60%) within
5
the duration of administration (from 4 weeks up to 1 year).102 How-
ever, the disease phenotypes induced by these species vary. In mice,
HFD usually results in an MetS phenotype characterized by obesity,
dyslipidaemia, hypertension and glucose intolerance, which could be
due to species-specific susceptibility to dietary fat.79,80,103 Further-
more, age has been shown to be an important factor modulating sus-
ceptibility to diet-induced MetS.104 In fact, compared with young
mice, old C57BL/6 mice appear to be more susceptible to HFD,
resulting in an aggravated phenotype.105 In rats, different outcomes
were observed according to the age at which the animals were first
exposed to HFD. In fact, Sprague–Dawley rats exposed to HFD at
3 weeks of age for 8 weeks developed obesity, impaired FBG,
dyslipidaemia and hypertension; however, when rats aged 8 weeks
were fed HFD, they only developed obesity and hypertension.61
Nonetheless, in Wistar rats, 8 weeks of HFD with 50% kcal from fat
were sufficient to induce components of MetS, such as obesity,
dyslipidaemia, impaired glucose tolerance (IGT) and hepatic
steatosis.106
Interestingly, HSD induces the same MetS phenotype in both
mice and rats, although most studies have been performed in the lat-
ter.107 Obesity often does not develop under this condition, although
different sources of sugar (sucrose vs. glucose vs. fructose) are added
to the diet.42,108 However, the absence of differences in BW does not
indicate identical body composition, as HSD-fed animals generally
show a significantly higher percentage of epididymal fat weight than
the controls.109–111 Furthermore, under this diet, both species
showed clear dyslipidaemia (hypertriglyceridaemia and hyper-
cholesterolaemia), accompanied by increased glucose intolerance and
insulin resistance, while presenting merely “mild” hypertension or
even the absence of BP elevation.112
Finally, the combination of high-fat and high-fructose diet
(HFHFD) is mandatory for achieving hyperglycaemia, which is another
feature of MetS. In 10-week-old Wistar rats fed HFHFD, impaired
fasting glucose (IFG) appears after 2 months of diet, whereas hyper-
glycaemia only manifested consistently after 6 months, and sustained
hyperinsulinaemia only after 8 months.74 In addition, in Wistar rats,
fructose supplementation via HFD (22.5% kcal from fat) was key to
aggravation of hyperglycaemia.113 In mice, one study demonstrated
that the supplementation of sugar to HFD worsened the disease phe-
notype via aggravation of the inflammatory state.114 Overall, HFHFD
exacerbated the MetS phenotype in both species, characterized by
overt/sustained fasting hyperglycaemia, dyslipidaemia, hypertension
and obesity.
2.3 | Prediabetes
In conjunction with the epidemics of T2DM, the prevalence of
prediabetes has skyrocketed worldwide, mainly due to major changes
in lifestyle associated with economic growth; in particular, altered
dietary patterns are promoters of obesity.115 Prediabetes is an
increasingly common asymptomatic condition associated with a high
risk for T2DM development.115,116 Prediabetes, or intermediate
 6

TABLE 1 Main features of diet-induced rodent models of metabolic syndrome

Diet Duration
type Composition (%) (weeks) Specie/strain Main features Refs
High-fat based
HFD 60% kcal from fat, 20% kcal from protein, 20% kcal 13 Male C57BL/6 mouse Obesity: 74% Δ weight gain; fivefold increase in fat Roopchand et al.78
from CHO (6-week-old) mass
Increased serum TGs
Elevated FBG: 350 mg/ml
IGT
60% kcal from fat, 20% kcal from protein, 20% kcal 20 Male AKR/J mouse (6- to Obesity: 43% Δ weight gain Meng et al.79
from CHO 12-week-old) IGT
Elevated right ventricle systolic BP
49% kcal from fat, 14% kcal from proteins, 37% kcal 8 Male Wistar rats (10- to Obesity: 26% Δ weight gain; 1.5-fold increase in Lasker et al.106
from CHO 12-week-old) epididymal fat mass
Increased plasma TGs
Decreased plasma HDL
IGT
Hepatic steatosis and inflammation
60% kcal from fat, 20% kcal from protein, 20% kcal 8 Male Sprague–Dawley rats Obesity: 141% Δ weight gain; 1.5-fold increase in Cheng et al.61
from CHO (3-week-old) rWAT mass
Increased plasma TGs
Decreased plasma HDL
Fasting blood glucose: 117.5 mg/dl
Increased systolic blood pressure
Hepatic steatosis
High-sugar based
HSD 16.9% kcal in fat, 18.1% of kcal in protein 65% of kcal 12 Male C57BL/6 (8-week-old) Obesity: no BW changes, slight increase in Do et al.42
in CHO (85% from glucose or fructose and 15% epididymal WAT mass
from sucrose) Increased serum cholesterol and LDL fraction.
FBG: 175 mg/dl
IGT
Hepatic steatosis
10% kcal from fat, 18.6% kcal from protein, 60.6% 16 Male Wistar rats (6-week-old) Obesity: no BW changes Aguilera-Mendez et
kcal from CHO + 30% fructose (w/v) in water Increased serum triglycerides al.111

Decreased serum HDL

Increased systolic BP
IGT
PREGUIÇA ET AL.
 PREGUIÇA ET AL.

TABLE 1 (Continued)

Diet Duration
type Composition (%) (weeks) Specie/strain Main features Refs
Hepatic steatosis

High-fat/high-sugar based
HSHFD 59% kcal from fat, 15% protein, 26% kcal from 8 Male C57BL/6 (12-week-old) Obesity: 62% Δ weight gain; threefold increase in Masi et al.114
CHO + condensed milk (23% kcal from fat, 9% kcal visceral adipose tissue
from protein, 68% kcal from CHO) Increased TGs
FBG: 214 mg/dl
Mild hepatic steatosis
60% kcal from fat, 20% from protein, 20% from 8 Male Sprague–Dawley rats Obesity: 26% Δ weight gain; 0.75-fold increase in Cheng et al.61
CHO + 30% sucrose (w/v) in water (8-week-old) rWAT mass
Elevated plasma TGs
Decreased plasma HDL
FBG: 99.7 mg/dl
Increased systolic BP
IGT
Early stage hepatic steatosis
22.5% kcal from fat, 17% kcal from protein, 42.3% 20 Male Wistar rat (9-week-old) Obese: 30% Δ weight gain; 1.84-fold increase in Moreno-Fernandez et
kcal from CHO + 25% glucose (w/v) in water epididymal mass al.113
Increased plasma TGs
Decreased HDL
FBG: 290 mg/dl
7
8 PREGUIÇA ET AL.
hyperglycaemia (the preferred terminology of WHO), defines a state
in which the individuals have abnormal glucose homeostasis where
blood glucose levels are above the normal but below the thresholds
established for T2DM diagnosis.115,117
The current gold standard measures used for prediabetes screen-
ing are FBG test and a 2-h plasma glucose analysis during an oral glu-
118
cose tolerance test (OGTT). Patients with prediabetes may have
IFG and/or IGT, which are defined by the American Diabetes Associa-
tion (ADA) as FBG levels of 100–125 mg/dl and 2-h glucose level of
140–199 mg/dl during an OGTT.119 Glycated haemoglobin (HbA1c)
concentrations varying from 5.7% to 6.4% is further considered by
ADA as an additional criterion for prediabetes diagnosis (Figure 1).119
Whereas IFG is mainly related to hepatic insulin resistance, IGT typi-
cally reflects peripheral insulin resistance and altered β-cell function.
Both insulin resistance and impaired β-cell function are responsible
for the increased risk of T2DM development in patients with
prediabetes.120–122
Unlike T2DM, options for prediabetes animal models are limited,
and unified classification criteria for rodents are lacking, for whom the
prediabetic phenotype has been defined/established according to the
following parameters: increased 2-h blood glucose values, glucose
intolerance, hyperinsulinaemia, hyperlipidaemia and moderate/mild
75
hyperglycaemia (Figure 1). Some diet-induced animal models have
been described to mimic the stages of prediabetes and/or insulin
resistance. Several experimental studies have shown that dietary mac-
ronutrient composition is an important environmental determinant of
the quality of insulin action and a trigger for the prediabetes
74,75,123
stage. Indeed, the modern WD and CD, rich in saturated fats
and in CHO (such as fructose and sucrose), are able to promote impor-
tant changes in CHO metabolism, causing prediabetes, although other
metabolic features resembling obesity and MetS may coexist.
Long-term HFD intake in several strains of rat and mouse can
cause hyperlipidaemia, peripheral insulin resistance, moderate hyper-
glycaemia and IGT, together with increase in the expression of inflam-
124,125
mation and oxidative stress markers. In rodents on HFD
feeding, insulin resistance is followed by insufficient β-cell compensa-
tion, resulting in hyperinsulinaemia and altered glucose metabo-
lism.126 The effects of HFD depend not only on the source and
percentage of calories but also on the animal species and strains
used.127–129
A “humanized” model of prediabetes was suggested for
young male Wistar rats under HFD (45% calories from fat) for
6 months.75 The authors reported a progressive development of the
phenotype typical of humans, starting with BW gain, gradual increase
of FBG and glucose intolerance, further evolving to hyperinsulinaemia
and insulin resistance, together with hypertrophy of pancreatic β-cells
in the last month.75 Increased levels of FBG and insulin and aug-
mented homeostatic model assessment of insulin resistance (HOMA-
IR), together with impaired β-cell function and OGTT, were also
observed in Sprague–Dawley rats under HFD (46% calories from fat)
for 12 weeks.130 As animal strains might present distinct responses to
HFD, the metabolic effects of HFD intake during 16 weeks were com-
pared between Wistar and Sprague–Dawley rats; results showed that
the majority of the metabolic changes (such as BW gain, fasting insulin
and leptin levels, HOMA-IR index and glucose intolerance) were
detected earlier and were more pronounced in Wistar rats.60 A robust
mouse model for IGT and early T2DM was established using female
C57BL/6J mice under HFD (58% calories from fat) for 12 months.131
One week after starting the diet protocol, the animals presented ele-
vated baseline glucose and insulin levels and IGT, along with impaired
insulin response; over time, progressive worsening of insulin resis-
tance was observed in HFD-fed mice.131 Another study performed in
12-week-old male C57BL/6J mice during a period of 10 weeks
showed that diets rich in both saturated (60% calories from fat, con-
taining lard) and monounsaturated (60% calories from fat, such as
from olive oil) fatty acids induced marked insulin resistance with
hyperinsulinaemia, accompanied by inflammation and altered distribu-
tion of adipose tissue.129
Several experimental studies have shown the use of HSD (mainly,
high-sucrose and high-fructose) as an inexpensive nutritional strategy
to promote the development of insulin resistance and glucose intoler-
ance in animal models; however, the results were inconsistent, appar-
ently due to variations in the metabolic responses to diets based on
species/strain, duration of protocol, percentage of sugar and route of
administration.77,132–135 In fact, a study suggested that C57BL/6 mice
are more resistant to sucrose treatment than Wistar rats.132 In that
study, male Wistar rats and C57BL/6 mice, both with 7 weeks of age,
received 30% sucrose in drinking water during 20 weeks. The mice
presented slight obesity and hyperlipidaemia without changes in glu-
cose and insulin levels, whereas rats presented hypercholesterolaemia,
hyperglycaemia, hyperinsulinaemia and insulin resistance.132 Two
studies using the same species and strain (male C57BL/6J mice), but
distinct sucrose content and protocol duration, reported the absence
of weight gain but diverse metabolic responses.77,84 In fact, 8 weeks
of HSD treatment (10% fat, 32% sucrose) of 3-month-old mice
resulted in adipocyte hypertrophy, glucose intolerance, hyper-
insulinaemia, hyperlipidaemia, hepatic steatosis and increased secre-
tion of inflammatory cytokines,77 whereas 4-week-old mice on
55 weeks of solid food with high sucrose (50%, wt/wt) content did
not show changes in BW, but variable degrees of hyperglycaemia and
hyperinsulinaemia.84 Furthermore, a study using male C57BL/6J mice
(8 weeks old) suggested that distinct modes of sucrose delivery differ-
entially affected body regulation and glucose homeostasis.134 The
authors showed that mice exposed to a liquid sucrose solution (50%
sucrose in the water plus 30% calories from sucrose in solid food)
presented higher adiposity, impaired glucose homeostasis, peripheral
insulin sensitivity and insulin resistance than mice maintained under
equivalent levels of sucrose in solid diet (73% calories from
sucrose).134 Previously, we were able to reproduce a prediabetic rat
model using an oral regimen of 35% sucrose consumption for
9 weeks.112,136 The animals were characterized to be prediabetic
based on fasting normoglycaemia, IGT, hyperinsulinaemia and insulin
resistance without obesity, which is consistent with the results of
other studies;137 interestingly, the animals concomitantly displayed
some early features of retinal and neuronal impairment.137–139
The use of fructose-rich diet is also a well-characterized approach
for eliciting insulin resistance and IGT in rodents.140,141 Unlike
PREGUIÇA ET AL.
glucose-fed rodents, fructose-fed rodents displayed disruption of
metabolic homeostasis, including IGT and dyslipidaemia.142,143 In one
study, low fructose (7%) content in drinking water was sufficient to
cause glucose intolerance with abnormal morphology and function of
pancreatic islet cells in Wistar rats, but without changes in FBG, insu-
lin and lipids levels.144 However, in another study, the same strain of
rats fed with fructose-rich diet (10% w/v in drinking water) developed
hypertriglyceridaemia and a state of insulin resistance (demonstrated
by hyperinsulinaemia with normoglycaemia, high insulin:glucose molar
ratio and HOMA-IR index) after 3 weeks of fructose
145
administration.
HFHSD is also commonly used to experimentally replicate predia-
betes.146,147 Different sources and amounts of CHO (mainly fructose
or sucrose) and fats have been used.148–150 In the HFHSD regimens
administered to rodents, CHO content typically ranged between 10%
and 60% (in the diet, drinking water or both), whereas fat content var-
ied between 20% and 60%.148,151 C57BL/6J mice fed HFHSD (45%
energy from fat and 20% by weight from sucrose) for 22 weeks
showed increased BW, glucose intolerance, fasting hyperinsulinaemia
and dyslipidaemia.146 Paradoxically, when the same diet was continu-
ously administered for additional 20 weeks, long-term (42 weeks)
HSHFD consumption was able to change glucose tolerance and
decrease plasma FFA levels, although the mice displayed elevated
FBG levels and dramatic hyperinsulinaemia.146 In Wistar rats, a predi-
abetic stage was achieved by feeding the animals during 8 weeks with
WD (containing 45% kcal from fat and 35% kcal from CHO) and 20%
sucrose solution in drinking water. The animals developed obesity,
mild hyperglycaemia, hyperinsulinaemia, hyperlipidaemia and glucose
intolerance.152 In other recent study, Vatandoust et al. 153
showed
that consumption of a combination of solid HFD (30%) with fructose
in drinking solution (30%) for 10 weeks effectively elicited a predia-
betic state in Wistar rats, which was confirmed by increased FBG and
TG levels, together with insulin resistance and decreased insulin
secretion from pancreatic β-cells.
2.4 | Overt diabetes
Compared with what occurs in prediabetes and according to ADA and
WHO, the current diagnostic criteria for diabetes are based on FBG
or 2-h plasma glucose levels during OGTT and on HbA1c levels
(Figure 1). A patient with diabetes is diagnosed when the FBG after
8 h of fasting is ≥126 mg/dl (7.0 mmol/L) or 2-h plasma glucose level
is ≥200 mg/dl (11.1 mmol/L) during an OGTT (using a glucose load
containing the equivalent of 75-g glucose solution) or HbA1c ≥ 6.5%
(48 mmol/L).119,154
Similar to the human condition, an animal model of T2DM relies
on the assessment of the key endpoints for glucose homeostasis,
namely, blood glucose and insulin levels, GTT, insulin tolerance test
(ITT), HOMA-IR, quantitative insulin sensitivity check index (QUICKI),
32,155–157
HbA1c and whole-body insulin sensitivity. In most preclinical
studies, FBG is the most common readout of a rodent's T2DM condi-
tion. However, standardized guidelines and completely unified
9
classification criteria for T2DM rodent models are lacking, probably
because normal and diabetic FBG differ considerably between rodent
species (Figure 1). It is generally assumed that the criteria used for rats
are closest to those used for humans, namely, FBG > 126 mg/dl
(7.0 mmol/L).158,159 In contrast, the basal levels of FBG in mice are
higher than those in rats and humans (ranging between 140 and
220 mg/dl), indicating that according to the aforementioned criteria,
all mice would develop diabetes. Hence, FBG values ≥250 mg/dl is
the recommended threshold for defining T2DM in mice.160,161 In addi-
tion, increased glycaemia following 2 h of OGTT frequently corrobo-
rates the FBG values. Here, the large discrepancy in the currently
reported FBG cut-off values is possibly due to not only to the animal
strain used but also to the degree of disease severity/duration and
the duration of the fasting period.162,163
Several different animal models of T2DM sharing many pheno-
typic features with the human disease are currently available,38,154
which includes genetically manipulated (spontaneous or trans-
genic/knockout), surgically treated (partial pancreatectomized animals)
and chemically induced (with STZ injection, for instance) ani-
mals.164,165 However, diet manipulations aimed at mimicking human
overnutrition and inadequate nutrient intake are often used (alone or
in combination with genetic/chemical approaches) to more closely
replicate the pathophysiological processes underlying T2DM progres-
sion (Table 2). Owing to the large heterogeneity of experimental pro-
tocols aimed towards establishing an accurate diet-induced T2DM
animal model, careful choice of the most adequate model for a spe-
cific purpose is required.
The separate intake of HFD or HSD can promote important
changes in metabolic pathways responsible for insulin resistance and
initial metabolic features underlying obesity, MetS or prediabetes, as
mentioned above. However, the use of diets enriched with fat or sugar
to induce overt T2DM in rodents has yielded conflicting results.166–170
170 For instance, C57BL/6J mice with a genetic predisposition for
developing features of T2DM have been widely used as diet-induced
models of T2DM and subsequent complications.171 In fact, although
some of the early diabetic features were noted immediately after
4 weeks, a longer duration (16–25 weeks) of HFD (40–60% of calories
from fat) allowed the development of a well-established T2DM pheno-
type, namely, higher hyperglycaemia, with FBG  240 mg/dl, IGT and
insulin resistance, along with adipose tissue inflammation and obe-
sity.172 This pattern of T2DM phenotype evolution in C57BL/6J mice
has been described in several reports.171,173,174
Unlike mice, the most commonly used rat strains (such as Wistar
and Sprague–Dawley) are not genetically predisposed for T2DM phe-
notype development.175 Nevertheless, there are noteworthy excep-
tions, including the male Zucker diabetic fatty (ZDF) rat, a well-
established genetic model of obese T2DM derived from the selective
inbreeding of obese Zucker (leptin receptor-deficient) rat.176 The male
ZDF rat displays a phenotype of progressive hyperglycaemia, reaching
a state of overt diabetes that can be accelerated or intensified with
HFD intake.177,178 Apart from the expenses, the main drawback of
this widespread model is the monogenic feature related to the muta-
tion of the leptin receptor, which is uncommon in humans, thereby
 Main features of diet-induced rodent models of prediabetes and overt diabetes
10

TABLE 2

Diet type Composition (%) Duration (weeks) Specie/strain Main features Refs
Prediabetes
High-fat based
HFD 46% kcal from fat, 24% kcal from 12 Male Sprague–Dawley rats Obesity: data not shown Abdel-Hamid and
CHO, 20.3% kcal from protein (±8-week-old) Increased FBG: ≈115 mg/ml Firgany130

Hyperinsulinaemia
IGT
45% kcal from fat, 35% kcal from 17 Male Sprague–Dawley rats Obesity: 32.2% Δ weight gain; Marques et al.60
CHO, 20% kcal from protein (7-week-old) 21.6% increase in fat mass
IGT
45% kcal from fat, 35% kcal from 17 Male Wistar rats Obesity: 66.9% Δ weight gain; 43% Marques et al.60
CHO, 20% kcal from protein (7-week-old) increase in fat mass
Fasting hyperinsulinaemia (at Week 9)
Increased HOMA-IR index (at Week 9)
IGT (at Weeks 9 and 17)
Hypertriglyceridaemia
59% kcal from fat, 20% kcal from 40 Male Wistar rats Obesity: 98% Δ weight gain; Chalkley et al.175
CHO (8-week-old) twofold increase in total fat mass
Mild elevated FBG: ≈135 mg/ml
Hyperinsulinaemia
Mild glucose intolerance
High-sugar based
HSD 35% sucrose in water standard 9 Male Wistar rats Obesity: no BW changes Nunes et al.112 and
diet (16-week-old) Fasting normoglycaemia: 102.90 mg/dl Burgeiro et al.136

Increased fed glucose: 162.90 mg/dl

Fasting hyperinsulinaemia
Increased HOMA-IR index
Decreased insulin sensitivity
IGT
Hypertriglyceridaemia
7% fructose (w/v) in water 12 Male Wistar rats Obesity: no BW changes Miranda et al.144
(6-week-old) Fasting normoglycaemia: 70.5 mg/dl
IGT
r-
PREGUIÇA ET AL.
TABLE 2 (Continued)

Diet type Composition (%) Duration (weeks) Specie/strain Main features Refs
Loss of β-cells
PREGUIÇA ET AL.

Increased β-cell mass

10% fructose (w/v) in drinking water
High-fat/high-sugar based
HSHFD 42% kcal from fat %, 37% kcal
from CHO, 21% kcal from protein,
45% kcal from fat, 35% kcal from
CHO; 21% kcal from protein
30% kcal from fat and 70% kcal from
CHO + 30% (w/v) of
fructose solution
Overt diabetes
HSHFD 24% kcal from fat + 25% (w/v)
fructose in water
3 Male Wistar rats Obesity: no BW changes Fracini et al.145
(±8-week-old) Fasting normoglycaemia: 90 mg/dl
Fasting hyperinsulinaemia
Hypertriglyceridaemia
22 Male C57BL/6 mice Obesity: ≈80% Δ weight gain; Kowalski et al.146
(8-week-old) increased fat mass
Fasting normoglycaemia: 189 mg/dl
Fasting hyperinsulinaemia
Impaired glucose tolerance
42 Obesity: ≈60% Δ weight gain;
increased fat mass
Fasting normoglycaemia: 160 mg/dl
Fasting hyperinsulinaemia
8 Male Wistar rats Obesity: 26% Δ weight gain; almost van den Brom et al.152
(4-week-old) equal to twofold increase for both
epididymal and perirenal fat weight
Elevated FBG: 192 mg/dl
Decreased insulin sensitivity
IGT
Hypertriglyceridaemia
10 Male Wistar rats Obesity: 23% Δ weight gain Vatandoust et al.153
(6-week-old) Elevated fasting glucose: 105 mg/dl
Impaired glucose tolerance
Hypertriglyceridaemia
16 Male Wistar rats Obesity: 23% Δ weight gain Van der Werf et al.159
(7-week-old) Elevated FBG: 135 mg/dl
Increased HOMA index
Fasting hyperinsulinaemia
Insulin resistance
Dyslipidaemia
11

(Continues)
 12

TABLE 2 (Continued)

Diet type Composition (%) Duration (weeks) Specie/strain Main features Refs
HFD 60% kcal from fat, 20% kcal from 21 Male C57BL/6J mice Obesity: 85% Δ weight gain; 20% Guo et al.172
CHO, 20% kcal from protein, (4-week-old) increased body fat mass
Elevated FBG: 237.9 mg/dl
Fasting hyperinsulinaemia
Elevated HbA1c
Increased HOMA indexes
IGT
Insulin resistance
HSHF + 30% kcal from fat and 70% kcal 12 Male Wistar rats Obesity: 23% Δ weight gain Vatandoust et al.153
STZ from CHO + 30% (w/v) for fructose (6-week-old) Elevated FBG: 170–198 mg/dl
solution + STZ (30 mg/kg, twice)
Elevated fed glucose: ≥340 mg/dl
IGT
25.7-wt% lard and 46.5-wt% f 56 Male Wistar rats Obesity: No Δ weight gain Barriere et al.191
ructose + STZ (25 mg/kg, thrice) (10-week-old) Elevated fasting glucose: 270 mg/dl
(at 42 weeks)
IGT (starting at 10 weeks)
Dyslipidaemia
Pancreatic β-cell dysfunction with
subsequent insulinopenia
HFD + High fat diet (undisclosed 12 Male C57BL/6 mice Obesity: 20% Δ weight gain Goodzari et al.188
STZ composition) + STZ (45 mg/kg) (8-week-old) Elevated FBG: 195 mg/dl
IGT
Hyperinsulinaemia
Dyslipidaemia
Insulin resistance
PREGUIÇA ET AL.
PREGUIÇA ET AL.
educing the translational relevance.179 Although some studies have
shown typical features of overt diabetes in rat models of diet-induced
T2DM, particularly with long-term protocols, the issue remains con-
troversial, as diverse results have been reported. Upon dietary supple-
mentation of fructose (25% in drinking water) within an HFD regimen
of 4 months, Wistar rats displayed T2DM features comprising sys-
temic hyperglycaemia (FBG > 126 mg/dl), insulin resistance (HOMA-
IR > 2.4), glucose intolerance, dyslipidaemia, oxidative stress associ-
159
ated with endothelial dysfunction and hepatic steatosis. Similarly,
Kalaki-Jouybari et al.180 also reported a diabetic rat model in Wistar
rats after supplementation with HFHSD (30% fat and 20% fructose)
for 5 months. These authors confirmed the presence of T2DM fea-
tures, such as hyperglycaemia (FBG > 200 mg/dl), insulin resistance
(elevated HOMA-IR), dyslipidaemia and hepatic steatosis.180 How-
ever, both studies confirmed the diabetic phenotype in animals dis-
playing FBG > 126 mg/dl, without discriminating between the time
period of fasting. On the contrary, several studies reported that rats
do not become overtly diabetic, even with extended periods of HFD
132,175 175
consumption. Accordingly, Chalkley et al. showed that the
long-term (10 months) intake of an HFD (59% of the calories as fat
plus 20% of the calories as carbohydrate) by Wistar rats induced obe-
sity with large amounts of central abdominal fat and insulin resistance.
Nevertheless, this protocol failed to replicate overt fasting hyper-
glycaemia.74,175 These observations were corroborated by other
authors who were unable to establish an overt T2DM phenotype after
chronic HFHSD administration in rats.123,181
As diet-induced paradigms for establishing overt T2DM are long-
lasting and highly demanding, STZ administration is widely used as a
tool for inducing partial β-cell destruction and impairment of insulin
secretion.164,182–184 This toxin is usually administered intraperitone-
ally (i.p.) via single or multiple injections of varying dosages, ranging
from 25 to 45 mg/kg BW in rats and 40 to 100 mg/kg BW in
185–189 183
mice. As an example, Mansor et al. showed that low doses
(15–25 mg/kg BW) of STZ in Wistar rats, following 3 weeks of HFD
feeding (60% calories from fat), induced a T2DM phenotype, whereas
30 mg/kg BW of STZ more closely recapitulated type 1 diabetes.
Identical results were recently reported by Guo et al.184 Conversely,
Srinivasan et al.190 reported that a single injection of 35 mg/kg BW of
STZ is the optimal dose for T2DM phenotype development in
Sprague–Dawley rats following 2 weeks of HFD (58% calories as fat),
with frank hyperglycaemia (FBG > 400 mg/dl) and hyper-
triglyceridaemia. Another report mentioned that HFHSD (30% and
70% of total calories from fat and carbohydrate, respectively) treat-
ment for 10 weeks, followed by injections of low doses of STZ
(30 mg/kg, in two steps with 1-week interval), led to T2DM in Wistar
rats.153 Furthermore, administration of HFHFD (containing 46.5-wt%
fructose and 25.7-wt% lard) combined with low-doses of STZ
(25 mg/kg, multiple injection) in Wistar rats for 56 weeks was used to
mimic the evolutive nature of T2DM and the underlying advanced
stage complications.191 At the end of the experimental protocol, the
animals displayed frank hyperglycaemia (>300 mg/dl), marked
dyslipidaemia, hepatic fibrosis and pancreatic β-cell dysfunction with
subsequent insulinopenia.191 Interestingly, in addition to signs of
13
cardiovascular injury, these animals developed microvascular
angiopathies, namely, nephropathy, retinopathy and behavioural signs
of peripheral neuropathy.191 In C57BL/6 mice, T2DM was success-
fully induced by HFD plus STZ (45 mg/kg) for 12 weeks.188 The ani-
mals presented increase in BW, and serum TG and total cholesterol
levels, as well as increased FBG (>150 mg/dl) and OGTT (>300 mg/dl)
responses, together with hyperinsulinaemia, IGT and insulin resis-
tance.188 In another study, the C57BL/6J mouse model of T2DM was
developed using HFD for 14 weeks with an STZ injection (100 mg/kg,
i.p.).192 Once again, these animals weighed significantly more than the
control mice and presented hyperglycaemia (FBG > 400 mg/dl) with-
out changes in TG levels.192 These observations have been corrobo-
rated by other reports.184,186,187
Finally, it should be emphasized that diet-induced regimens com-
bined with STZ injection should closely replicate the pathophysiologi-
cal mechanisms underlying T2DM progression. As high doses of STZ
(>50 mg/kg) rapidly lead to destruction of pancreatic β-cells with
acute insulin deficiency (a feature of typical type 1 diabetes), low
doses of STZ should be used as a more suitable approach for experi-
mental T2DM and associated complications.
2.5 | Nonalcoholic fatty liver disease
NAFLD is currently the most common chronic liver disease, with an
estimated global prevalence of 25%.193 NAFLD encompasses a spec-
trum of diseases that develop in the absence of excessive alcohol con-
sumption, ranging from simple steatosis, the hallmark feature of the
disease, to nonalcoholic steatohepatitis (NASH), a more severe form of
NAFLD characterized by inflammation and fibrosis. NAFLD and NASH
may progress to cirrhosis and ultimately hepatocellular carcinoma
(HCC).194 The development and progression of NAFLD is linked to vis-
ceral adiposity, insulin resistance, dyslipidaemia and T2DM, which in
turn are driven by several risk factors, including high intake of satu-
rated fats and/or sugar, sedentary lifestyle and genetic susceptibility.
The lack of approved pharmacological therapies for
NAFLD/NASH has stimulated intense drug discovery efforts in this
field. Several animal models of NAFLD have been developed, mostly
in mice and rats, as platforms for increasing our understanding of the
disease pathogenesis and for the evaluation of therapeutic interven-
tions. However, the currently available models have translational limi-
tations and do not completely recapitulate the entire spectrum of
human disease progression. Animal models of NAFLD use dietary,
chemical, genetic or a combination of multiple approaches to recreate
specific pathogenic features. The assessment of NAFLD/NASH in
rodents is commonly based on liver histological analysis, liver triglyc-
eride content and plasma biomarkers (Figure 1).
As the primary trigger of NAFLD is overnutrition leading to obe-
sity, rodent models based on nutritional manipulation are thought to
best reflect the human pathology. The majority of animal models focus
on providing dietary components individually or in combination to
induce simple steatosis and steatohepatitis (Table 3). Furthermore,
even in genetic animal models of NASH, diet acts as a secondary
14
trigger for disease progression. Dietary regimens with high fat content
(45–75% energy from fat) are widely used to induce NAFLD in
195
rodents. For example, an early study showed that consumption of
HFD for approximately 10 weeks led to obesity, insulin resistance,
hyperinsulinaemia, glucose intolerance and hepatic steatosis, whereas
increase in alanine aminotransferase (ALT) and aspartate aminotrans-
ferase (AST) levels, indicative of steatohepatitis, was observed only
after 34–36 weeks in male C57BL/6J mice.196 Another study reported
steatosis in Sprague–Dawley rats within 1–2 weeks after HFD
onset,197 whereas only minimal fibrosis was observed after extended
196,198
exposure (36–50 weeks). However, the results regarding the
onset and degree of steatosis, inflammation and fibrosis varied, which
appeared to depend on species, gender, dietary fat content and com-
position and duration of HFD feeding.195 Nevertheless, HFD more
closely resembles the metabolic parameters of human NAFLD,
although the pathological and molecular alterations are not as severe.
The methionine–choline-deficient (MCD) diet is a well-
established nutritional model of advanced NASH. The complete defi-
ciency of the essential nutrients, methionine and choline, combined
with high sucrose (40% of energy) and moderate fat (10–20%) con-
tent, results in impaired formation and secretion of very low-density
lipoprotein (VLDL) and impaired hepatic β-oxidation. This model is
characterized by macrovesicular steatosis, hepatocellular death, lobu-
lar inflammation, oxidative stress (after 3–5 weeks) and early onset of
liver fibrosis (after 8–10 weeks) in C57BL/6J mice.199 A limitation of
the MCD diet model is that its metabolic profile is opposite of that of
human NASH, as this model shows significant weight loss (up to 40%
in 10 weeks) and no peripheral insulin resistance, as well as low serum
insulin, fasting glucose and triglyceride levels,200 which prevents long-
term experimental fibrogenesis. These limitations can be overcome by
long-term feeding of a choline-deficient HFD (CD-HFD), which
induces the key features of MetS (obesity and insulin resistance),
steatosis, fibrosis and HCC development.201
The choline-deficient, L-amino acid-defined (CDAA) dietary model
is similar to the MCD model regarding choline deficiency, although
the dietary proteins are replaced by an equivalent and corresponding
mixture of L-amino acids. The CDAA model recapitulates features of
human NASH in rodents by sequentially inducing steatohepatitis, liver
202,203
fibrosis and HCC without any loss of BW. Feeding the CDAA
diet to rats results in rapid progression of steatosis to fibrosis,
followed by a rise in ALT level.203 In mice, the diet caused little or
negligible increase in ALT,204 and long-term feeding (20 weeks) was
202
required before liver fibrosis was observed. Finally, combining the
CDAA diet with HFD accelerated the development of NASH with
fibrosis (6–9 weeks) in mice.205
Western (atherogenic) diets typically contain cholesterol
(1–1.25%) and cholate (0.5%), which promote atherosclerosis,
resulting in liver damage and fibrosis in experimental animals. Male
C57Bl/6J mice fed WD (30% kcal from fat) exhibited steatosis, and
increased ALT and total cholesterol levels after 6 weeks, and hepato-
206
cellular ballooning and fibrosis after 24 weeks. However, obesity
was not observed in this model, and glucose tolerance and insulin sen-
sitivity appeared to improve.206 The addition of a high fat component
PREGUIÇA ET AL.
to the atherogenic diet (80% kcal from fat) promoted hepatic insulin
resistance and further accelerated the progression to NASH.206
Finally, male and female C57Bl/6 mice fed a diet with high fat (40%
kcal from fat), cholesterol (2%) and fructose (42 g/L) levels in the
drinking water for 25 weeks exhibited obesity, steatosis with hepato-
cyte ballooning and progressive fibrosis.207
In rodents, the addition of fructose to drinking water results in
steatosis without features of NASH after 8 weeks and induces a sig-
nificant increase in BW, and plasma TG and glucose levels.208,209
Recently, Asgharpour et al.210 developed a dietary mouse model
based on an inbred isogenic strain of C57BL6/J and S129S1/svlmJ
mice fed HFD with ad libitum consumption of glucose and fructose.
These mice sequentially developed steatosis (4–8 weeks),
steatohepatitis (16–24 weeks), progressive fibrosis (16 weeks
onwards) and HCC.210
3 | DISCUSSION
3.1 | Open challenges for diet-induced animal
models
In strict contrast to the expectations of the medical and scientific
community, research on animal models has not been translated into
significant therapeutic advancements, particularly for more complex
and multifactorial diseases, such as the obesity-related metabolic dis-
orders. One reason for this failure may be the inability to translate the
enormous preclinical research into effective therapies in humans,
which may be strongly related to the complexity of these diseases per
se, and also to the poor representativeness of animal models with
respect to human diseases.
Actually, in most of cases, the way the disease is induced, in par-
ticular, the main features of its evolution, do not reliably represent the
complex pathophysiology associated with the development of obesity,
its evolution to MetS and prediabetes and further progression to
T2DM, with complications and associated consequences, including
NAFLD. Hence, a large number of studies using animal models are
induced by hypercaloric diets, which in some ways can more closely
mimic the evolution of diseases in humans. In fact, unhealthy eating,
typically hypercaloric diets, based on high sugar and fat contents and
low amounts of micronutrients, is one of the main risk factors (and
direct contributors) for the appearance and evolution of these condi-
tions. Furthermore, diet-induced models are usually associated with
slow evolution, thus imitating human disease progression and opening
up the possibility of testing the efficacy of preventive or curative ther-
apeutic or nutraceutical strategies.
Notwithstanding these advantages, the use of animal models of
obesity and related metabolic diseases induced by hypercaloric diets
is not free from limitations. On the contrary, they are usually associ-
ated with variables that strongly affect the type of model that can be
obtained, as discussed below. Another important issue involves the
precise definition and application of criteria that enable reliable
cataloguing of a model. Although criteria for human diseases are
TABLE 3 Main features of diet-induced rodent models of NAFLD

Diet type Composition (%) Duration (weeks) Specie/strain Main features Refs
High-fat based
PREGUIÇA ET AL.

HFD 60% kcal from fat (lard, soybean oil), 50 Male C57BL/6J mice (8-week-old) Hepatic steatosis without inflammation Ito et al.196
20% kcal CHO, 20% kcal from protein (10 weeks)
Steatohepatitis with inflammatory
markers (40 weeks)
Obesity: 83% Δ weight gain
Glucose intolerance
Insulin resistance
Hyperinsulinaemia (10 weeks)
42% kcal from fat (lard, corn oil), 16 Female Sprague–Dawley rat Hepatic steatosis Gauthier et al.197
36% kcal from CHO, 22% kcal protein (6-week-old) Obesity: 26% Δ weight gain; 1.4-fold
increase in fat mass
Elevated plasma glucose: 153 mg/dl
Hyperglycaemia (12 weeks)
Hyperinsulinaemia
Hypertriglyceridaemia
45% kcal from fat (lard, soybean oil), 43 Male Sprague–Dawley rat Hepatic steatosis with inflammation Omagari et al.198
35% kcal from CHO, 20% kcal (3-week-old) and mild fibrosis
from protein Obesity: 188% Δ weight gain
Elevated FBG: 174 mg/dl
Hyperinsulinaemia
Methionine and choline deficient diets
MCD Methionine-choline deficiency, 8 Male C57BL/6J mice Hepatic steatosis (3 weeks), McCuskey et al.199
sucrose (40%) and fat (10%) (8-week-old) necroinflammation (5 weeks) and
perisinusoidal fibrosis (8 weeks)
CD-HFD Choline deficiency, and high fat (45%) 52 Male C57BL/6J mice Mild hepatic steatosis (12 weeks), Wolf et al.201
(4- to 5-week-old) marked steatosis (24 weeks),
hepatocyte ballooning and fibrosis
(52 weeks)
Obesity: 75% Δ weight gain; more than
threefold increase in fat mass
Dyslipidaemia
IGT (24 weeks)
CDAA Choline deficiency, L-amino 16 Male Fischer 344 rat Hepatic steatosis and inflammation Uto et al.203
acid-defined (7-week-old) (2 weeks)
Hepatic fibrosis and cirrhosis
(16 weeks)
15

(Continues)
 (Continued)
16

TABLE 3

Diet type Composition (%) Duration (weeks) Specie/strain Main features Refs
Elevated blood glucose: ≈140 mg/dl

Hyperinsulinaemia
CDAA Choline deficiency, L-amino acid-defined 22 Male C57BL/6J mice (8-week-old) Hepatic steatosis with inflammatory Miura et al.202
(14% kcal from fat, 68.5% kcal from CHO, cell infiltration, hepatocyte
17.4% kcal from protein) ballooning and death, and hepatic
fibrosis
Obesity: 45.6% Δ weight gain; more
than threefold increase in epididymal
fat mass
Increased HOMA-IR index
Insulin resistance
Hypertriglyceridaemia
CDAA-HFD Choline deficiency, L-amino acid-defined, 14 Male C57BL/6J mice (6-week-old) Hepatic steatosis with lobular Matsumoto et al.205
high fat, methionine (0.1%) (62% kcal from inflammation and hepatocyte
fat, 21% kcal from CHO, 18% kcal from ballooning (3 weeks), hepatic
protein) fibrosis (6 weeks)
Obesity: 30% Δ weight gain
Hypertriglyceridaemia
Western (atherogenic) diet
WD Cholesterol (1.25%), cholate (0.5%) 24 Male C57BL/6J mice (8-week-old) Hepatic steatosis and inflammation Matsuzawa et al.206
(6 weeks), hepatocellular ballooning
and fibrosis (24 weeks)
Obesity: −4% Δ weight gain in relation
to control
FBG: 85 mg/dl
Hyperinsulinaemia
Increased cholesterol
WD+HF Cholesterol (1.25%), cholate (0.5%), 24 Male C57BL/6J mice (8-week-old) Exacerbated hepatic steatosis and Matsuzawa et al.206
high fat (60%) inflammation (6 weeks)
Hepatocellular ballooning and fibrosis
(24 weeks)
Obesity: −12% Δ weight gain in
relation to control
FBG: 93 mg/dl
Hyperinsulinaemia
Increased cholesterol
High-sugar based
PREGUIÇA ET AL.
 PREGUIÇA ET AL.

TABLE 3 (Continued)

Diet type Composition (%)
HSD 20% (w/v) fructose in drinking water
30% (w/v) fructose in drinking water
Combinations
HSHFD 40% kcal from fat and 2%
cholesterol + 42%, (w/v) of
fructose
42% kcal from fat, 0.1% kcal from
cholesterol + solution of 23%
fructose and 18% glucose
Duration (weeks) Specie/strain Main features Refs
8 Male Wistar rats (8-week-old) Hepatic steatosis Mamikutty et al.208
Obesity: 6.9% Δ weight gain
FBG: 145.8 mg/dl
Hypertriglyceridaemia
Elevated systolic blood pressure
8 C3H/HeOuJ mice (6- to 8-week-old) Hepatic steatosis and increased Spruss et al.209
inflammatory markers
Obesity: 24.2% Δ weight gain
25 Male and female C57BL/6J mice Hepatic steatosis, hepatocyte Charlton et al.207
ballooning, fibrosis and lobular
inflammation
Obesity: 68% Δ weight gain
Elevated glucose levels: 243 mg/dl
Hyperinsulinaemia
Hypercholesterolaemia
52 Male B6/129 mice (8- to 12-week-old) Hepatic steatosis (4 weeks), hepatocyte Asgharpour et al.210
ballooning (8 weeks), steatohepatitis,
lobular inflammation and progressive
fibrosis (16 weeks), spontaneous
hepatocellular cancer (52 weeks)
Obesity: 50% Δ weight gain
Insulin resistance
Hypertriglyceridaemia
17
18
defined by reputable international medical–scientific organizations,
the same is not always true for animal models. An exemplary case is
that of obesity. In humans, waist circumference is recognized as a
good predictor of obesity, which has been consistently shown to pro-
vide independent and better information than BMI, despite limited
implementation in routine clinical practice.53 In addition, information
regarding the amount and distribution of adipose tissue is of potential
interest; however, it remains to be accurately assessed.52 In animal
models of obesity, weight gain values and/or quantitative or qualita-
tive changes in the various adipose tissue deposits that can be used to
universally define cut-offs for rodents (rat and mouse) are lacking. The
absence of well-established criteria conflicts with the scientific rigour
of these experiments, rendering the translation of preclinical results
difficult. As mentioned above in the article, a similar absence of clear
criteria applies to animal models of diet-induced diabetes.
3.2 | Issues to consider when choosing a diet-
induced animal model
When a research group proposes to work with diet-induced animal
models, it must consider all the variables that may interfere with the
PREGUIÇA ET AL.
model, as summarized in Figure 2. The choice of the model will
depend on the study purpose and the pathophysiological relevance of
the model. Animal models are used for various purposes, including
understanding the mechanism of disease pathogenesis, identification
of new biomarkers and/or evaluation of therapeutic options, among
others. Although the majority of genetic or diet-induced models of
metabolic diseases are obese, the presence or absence of obesity
depends on the study purpose. Furthermore, some diet-induced
models of obesity and related disorders evolve with other conditions,
including dyslipidaemia and vascular complications,12 which may be
essential for the study, or confounders, depending on the study
objectives.
The duration of the protocol is a strong determinant of the meta-
bolic outcome, as mentioned throughout this article. In general, con-
sidering the above noted variations between different species and
strains, obesity and MetS in rodents can be achieved by submitting
the animals to few (up to 10) weeks of HFD, although the percentage
of BW increase and the changes in fat composition and distribution
may vary substantially between studies, as described above. Overall,
HSD cannot induce robust models of obesity but can be used to
develop MetS and prediabetes.112,136 Reduction of protocols might
be possible, for example, when combining HFD with HSD, which
F I G U R E 2 Major variables to be considered
when choosing a diet-induced rodent model of
obesity and related metabolic disorders, with
focus on animal-related factors and protocol-
related factors. HFD, high-fat diet; HSD high-
sugar diet; MetS, metabolic syndrome; NAFLD,
non-alcoholic fatty liver disease; Ob, obesity; STZ,
streptozotocin; T2DM, type 2 diabetes mellitus
PREGUIÇA ET AL.
usually causes slight impairment of both glucose and lipid metabolism,
with obesity, increased TG and glucose intolerance as possible out-
comes. If longer protocols of HFD or HSD (but especially when com-
bined) are chosen, more advanced metabolic stages, such as NAFLD,
can be achieved in rodents. Overt diabetes, in particular, is easily (and
rapidly) achieved when STZ is used to accelerate β-cell failure. How-
ever, the associated toxicity introduces an additional variable. A sum-
mary of the most representative protocols for each condition is
shown in Tables 1, 2 and 3.
Regarding the animal variables, species and strain differences are
critical points when selecting a model, as the susceptibilities to DIO
and related metabolic disorders vary significantly in both rat and
mouse,62,63,211 as discussed earlier in the article. Hence, whenever
possible, more than one species and strain should be used. In fact,
numerous studies based on single strains have shown that a subset of
the most commonly used rodent strains, namely, the inbred C57Bl/6
mice and the outbred Sprague–Dawley rats, do not respond to HFD
feeding.68,212 Importantly, studies evaluating a large collection of
inbred strains not only provide better understanding of this variation
but also mimic the diversity observed in human populations. One such
study compared the metabolic responses to HFD in males and females
of 43 inbred mouse strains.213 That study reported broad phenotypic
213
diversity among strains and gender for most traits measured. It is
noteworthy that specific rat and mouse strains from different sup-
pliers differ significantly, which introduces an additional degree of
complexity to the issue.
These issues cannot be separated from genetic and epigenetic
diversity, as it is well known that obesity and associated disorders are
caused by a combination of environmental factors (such as diet) and
polygenic predisposition. Some inbred strains are maintained to spon-
taneously develop a DIO phenotype, which are crucial for determining
the precise underlying genetic mechanisms. Unfortunately, inbred
strains that are prone to DIO development represent only a small frac-
tion of human metabolic phenotypes.59,214 Recently, researchers have
focused on outbred strains, which represent the polygenic variability
of the human population more reliably.215 The administration of HFD
to an outbred strain (e.g., Wistar or Sprague–Dawley) will differently
impact the animal's metabolism; whereas some will show rapid
increase in weight (OP), others will not show any changes in BW
(OR) despite the environmental pressure.70 Thus, the first step in
these studies involves identification of the animals that present a
polygenic predisposition or resistance to obesity. The differences
between these phenotypes are the keys for understanding metabo-
lism and the mechanism via which genetics and the environment
interact and modulate each other. In fact, there are several character-
ized differences, namely, gastrointestinal fat absorption, appetite,
216
physical activity, fat storage and/or consumption. These changes
appear to indicate that these animals have contrasting metabolic flexi-
bility, a readout of their capacity to adjust fuel oxidation to fuel avail-
ability.217 In fact, studies have shown that different mice strains with
distinct genetic backgrounds may display significant differences in glu-
cose pathways, detected as early as from the time of weaning.218
Regarding fat storage, OP rats are characterized by the presence of
19
larger adipocytes and higher visceral adipose inflammation than their
OR counterparts.219 Gender differences were observed when studies
showed that male mice are more likely to become obese than female
mice and that the protection against obesity in female mice is obliter-
ated by ovariectomy.220 Other relevant female-specific physiological
conditions, such as menopause and breast cancer, have been revealed
via modelling of diet-induced obesity with OP/OR rats.221 This strat-
egy may also be useful for studying the mechanisms underlying the
development of obesity-related disorders, such as MetS, diabetes and
NAFLD.
The role of sexual dimorphism in the development of several types
of diseases, including obesity-related metabolic disorders, is an emerg-
ing issue that has triggered enormous interest in the scientific commu-
nity, and which cannot be overlooked when selecting animal models
of metabolic diseases.211,221,222 Sex must be considered a biological
variable as important, if not more, than other variables, such as age or
strain. For many years, experimental animal research has been per-
formed on males, with the argument that the female hormonal cycle
induces higher variability and that the metabolic disease phenotype is
more pronounced in males, However, obesity, T2DM and other asso-
ciated metabolic diseases occur in both men and women, despite
some important sex-based differences in susceptibility to DIO and
diet-induced insulin resistance.223,224 Although obesity is more preva-
lent among women,225 they are usually less likely to suffer from diet-
induced consequences of T2DM,226–228 which is in line with its lesser
global prevalence in women than in men.229 The effect of gonadal
hormones on fat distribution might explain, at least partially, the sex-
ual dimorphism observed in both women and female rodents. In fact,
compared with their male counterparts, females have less visceral and
more subcutaneous fat, which negatively affects cardiometabolic
risk.230,231 In female rats and mice, the masculinization of fat distribu-
tion due to ovariectomy, which ablates the production of endogenous
oestradiol and progesterone, augments the susceptibly to diet-
induced insulin resistance.232 Evidences suggest that sex chromo-
somes, independent of gonadal sex, are also involved in distinct meta-
bolic pathways, which may be linked with susceptibility to DIO and
diet-induced insulin resistance, such as feeding behaviour, glucose
homeostasis, adiposity and fatty liver.233,234
Nowadays, improved information on sex differences in rodent
species and strains are available, some of which have been obtained
from elegant studies using OP/OR or DIO/DR rodents selected from
outbred and/or inbred strains; these are indubitably valuable tools for
studying obesity and the associated metabolic complications, includ-
ing sex-specific differences.221,235,236 In general, progression of DIO is
comparable between male and female rats,68,237 whereas male mice
are more susceptible than females, developing obesity earlier and
more prominently.220,232 Sexual dimorphism regarding diet-induced
insulin resistance and glucose intolerance is also observed in both
mice and rats, with males being the most affected.237,238 The same
pattern is observed in most rodent models of T2DM.222 As described
above, the ZDF rat is a clear and interesting example, as males
develop severe T2DM under normal rodent chow diet, unlike the
inability of females to develop hyperglycaemia, despite developing
20
obesity to an extent similar to that observed in the males.177,239 The
overall conclusion is that the reliability of the results and their poten-
tial for translation into human disease-related research can signifi-
cantly increase if researchers consider sexual dimorphism when
selecting the animal model, as has been strongly suggested in previous
studies.222,240,241
Furthermore, animal age should be carefully considered during
the experiment because of its effect on the disease, in particular, the
age at the beginning of a dietary intervention. In humans, increasing
age is a risk factor for the development of chronic metabolic diseases.
BW increases with age, accompanied by redistribution of fat mass,
namely, to the muscle and hepatic tissues, which might explain the
increased susceptibility to glucose intolerance and insulin resistance,
as reviewed by Kleinert et al.10 Similar metabolic changes in BW and
242,243
fat composition and redistribution occur in rodents with age.
However, glucose intolerance in rats is affected more by age than in
10
mice. The age at the beginning of a dietary protocol should also be
considered, as metabolism changes substantially from preadult age to
old age, and different effects of dietary interventions should be
expected if young, adult, or old animals are used at the beginning of
61,211
the protocol.
The type of diet and its specific composition are also particularly
important. The specific composition of diets, such as the percentages
of CHO, fats and proteins (as well as fibre and water), considerably
affects the appearance and progression of obesity and associated con-
ditions and complications.244,245 Furthermore, researchers should also
consider the effect of the micronutrient composition on the study
outcome, as they are responsible for several key metabolic functions
in distinct organs and tissues.246–248 In fact, intake of vitamins and
mineral plays a key role in moderating the hyperphagic behaviour of
44
C57BL/6J mice fed HFD. The percentage of fat is a key aspect that
should be considered. Diets containing 45% and 60% fat by calories
are considered HFD for rodents, as both induce obesity. As mice on
60% fat diet become more obese in less time than mice on 45% fat
35
diet, the 60% rodent diet appears to be better at first. However,
compared with the typical American or European human diet (con-
taining about 30–40% fat by energy), experimental use of diets with
high levels of fat do not adequately mimic the levels of fat in the
human diet. A detailed analysis of the resultant obesity and metabo-
lism of mice fed 45% and 60% fat diets revealed that both the extent
of obesity and dysregulation of glucose homeostasis increased with
the fat percentage.103 Comparison of the metabolite profiles in the
lungs of mice fed low fat (10%) versus HFD (45% fat) versus very
HFD (60% fat) showed that the profile of 80 metabolites had changed
in the HFD-fed mice relative to that fed the control diet.249 Alter-
ations in the metabolites of the tricarboxylic acid cycle, antioxidants
and nucleotide pathways, as well as significant changes in neutral
lipids, fatty acids, phospholipids and sphingolipids, suggested that diet
composition and obesity induced lung metabolome changes, which
may contribute to disease progression.249 A caveat of this type of diet
is the absence of other components, such as fatty acids, which are
known to influence the level of obesity, as its presence in rodent diets
250
may better mimic human diet. In fact, the oxidation and deposition
PREGUIÇA ET AL.
rates vary with fatty acids, contributing to different responses regard-
ing fat accumulation, weight gain, insulin resistance and
244,250–253
inflammation. Whereas monounsaturated fatty acids posi-
tively affect weight control via increase in postprandial fat oxidation
and diet-induced thermogenesis, the intake of saturated fatty
acids251,252 or n-3 polyunsaturated fatty acids (PUFAs; flaxseed oil
rich in linoleic acid) produces opposite effects, promoting body fat
accumulation.250,253 Furthermore, the micronutrient components of
TWD have been used in rodent studies to evaluate their effect on
weight gain and biomarkers of metabolic function.45 This report
showed that mice fed HFD containing vitamins and oil minerals, which
reflects the Western dietary pattern, inhibited hyperphagia and
weight gain associated with the higher fat content of TWD. These
observations reinforce the importance and necessity of considering
the contribution of micronutrients intake in preclinical models of obe-
sity and associated metabolic disorders.45 It is noteworthy that some
transgenic or knockout models may not show an overt diabetic phe-
notype under normal conditions and have to be fed hypercaloric diet
to completely express the effect of genetic background on the
expected phenotype.254–256 In addition to diet composition, the tex-
ture and palatability of the diet pellets are important factors affecting
consumption, considering the high sensitivity of animals to sensorial
stimuli; this will ensure that diet consumption remains unchanged due
to these parallel factors.
Importantly, in studies on diet-induced rodent models of meta-
bolic disorders, the amount of food consumed should be accurately
monitored, as rodents fed more energy-dense diets (such as DIO 45
or 60) will compensate by consuming less food. However, despite
the lower intake, they will consume more energy than animals fed
lower energy feed (such as AIN-93). In addition, both total and
fractionated (from CHO, fat and protein) caloric intake should be
calculated, as they may be potentially relevant for the study. In
this context, the inclusion of accurate control groups fed diets of
composition that can be properly compared with those of the
diseased animals should be considered when planning an experi-
mental protocol.
Regarding the choice of proper control diets, most metabolic stud-
ies have frequently used refined diet (e.g., refined HFD or rHFD), in
which each nutrient is supplied by a specific and purified ingredient,
compared with a plant-based, unrefined control chow diet.32,257
However, refined and chow diets exhibit marked differences in
micronutrient and macronutrient content. Therefore, when comparing
the effects of a chow diet with those of a refined HFD, the effects of
the dietary fat will be confounded with the effects of other compo-
nents in the diets. The two major differences between chow and
refined diets are the phytoestrogen content, which is high in chow
diets but is absent from refined diets, and sucrose, which is used as a
carbohydrate source in refined diets but is absent from chow. In addi-
tion, chow diets are not standardized, as their composition varies
with the supplier and batch. Considering these differences, a refined
low-fat diet (rLFD), formulated using the same purified ingredients as
rHFD, is a more appropriate control diet than chow,258,259 which is
particularly clear when focusing on the effects of gut microbiota,260
PREGUIÇA ET AL.
as will be discussed later. However, recent studies have reported
conflicting results regarding the metabolic effects of LFD and chow
diets. In one study, mice fed LFD did not show changes in BW or fat
mass,261 which disagrees with the results of other studies in which
mice fed LFD showed increased BW and adiposity compared with
mice fed chow diet.258,259 Furthermore, the effects of rHFD on meta-
bolic outcomes, such as adiposity, inflammation and insulin sensitiv-
ity, depend on the control LFD used, which further reinforces the
notion that comparisons of studies using chow as the control diet are
prone to unintended but significant metabolic disturbances, strongly
suggesting that adequate rLFD should be used as the control for
rHFD.258,259
The manner in which determinations are performed (such as
fasted or fed conditions and tests, among other relevant details) and
the researchers' experience in performing these procedures should
also be considered. In this complex system, even the origin (supplier)
of the animals and their housing conditions (e.g., temperature) can
affect the results, for example, by interfering with gut microbial com-
munity of rodents, as discussed below. 262
In fact, housing temperature is another variable that profoundly
affects the outcome and interpretation of metabolic experiments. An
increasing body of evidence shows that the cold stress to which
rodents are commonly subjected significantly affects physiology and
may compromise the ability to model some human diseases, namely
obesity and related disorders.263,264 Most studies are conducted at an
ambient temperature of 20–22 C, which is below the thermoneutral
 265
zone for mice and rats (29–31 C). Housing of rodents below their
thermoneutral zone results in the activation of adaptive thermogene-
sis for maintaining their core temperature. Indeed, at 22 C, rodents
expend twice as much energy and consume more food than those at
 265
ambient temperatures of 30 C, which results is substantial differ-
ences in their metabolic, cardiovascular and immune phenotypes.
Thus, the constant thermal stress induced by the animal housing con-
ditions may significantly affect the outcomes of previous metabolic
studies. Certain phenotypes observed under conditions of thermal
stress disappear when mice are housed at thermoneutrality, whereas
others that are more consistent with human physiology emerge. For
example, a recent study showed that male C57BL/6J mice housed at
thermoneutrality showed accelerated onset of HFD-induced inflam-
mation in WAT and vasculature, which promotes atherosclerosis but
266
not insulin resistance. Another study using C57BL/6 mice investi-
gated the role of thermoneutral housing as opposed to standard hous-
267
ing in HFD-induced NAFLD. The authors reported that 24 weeks
of thermoneutral housing lowered stress-driven production of cortico-
sterone, increased proinflammatory immune responses and exacer-
bated HFD-induced NAFLD pathogenesis without significant changes
267
in BW gain. In particular, female mice housed at thermoneutrality,
which are typically resistant to HFD-induced obesity and NAFLD, also
developed severe obesity and hepatocellular damage.267 However,
others have challenged this notion and suggested that the optimal

ambient temperature is 23–25 C for single-housed mice and
20–22 C for group-housed mice.268 Nevertheless, future research in
thermoneutral rodents might enable more predictive modelling of
21
human diseases and might also yield important insights regarding our
understanding of mammalian physiology.
The gut microbiota plays major roles in host homeostasis due to
several metabolic functions associated with the regulation of glucose,
lipids and energy.269 Several lines of evidence suggest that impair-
ment of the gut microbiota (dysbiosis) contributes to the development
of obesity, diabetes and associated complications.270 At the same
time, the microbial composition is highly shaped by dietary substrates,
and HFD supply has been associated with changes in gut microbial
composition and diversity, often referred to as obese microbiota.271
Conversely, other studies reported body fat accumulation, hepatic
steatosis and fasting hyperglycaemia in animals fed standardized WD,
independent of gut microbiota dysbiosis,46 thereby indicating an
effect of specific diet composition on the gut microbiome. Fibres, in
particular, are chief dietary substrates shaping gut microbiota compo-
sition.272,273 Gut microbiota modulation using dietary components is
important in the context of both hypercaloric and control diets used
in animal models of obesity and associated metabolic disorders. In
fact, as mentioned above, preclinical studies often use “chow diet” as
the control for refined HFDs, which may result in significant bias, as
refined diets contain purified ingredients, whereas chow diets contain
dietary fibre obtained from unrefined cereals and legumes, such as in
low fat grain-based diet.260 The absence of fermentable fibres can
explain, at least partially, why mice fed refined low-fat diet (rLFD), for-
mulated using the same purified ingredients as rHFD, presented more
adiposity than chow-fed mice.259,260 These experiments emphasized
that chow diet and rLFD are indeed not nutritionally equivalent and
that the former should be used as the control for rHFD.258,259
Complementing these observations, Dalby et al.261 showed that
microbiome composition was more dependent on whether the experi-
mental diet was refined or complex, independent of an obese pheno-
type, suggesting more careful characterization of the association
between gut microbiota and obesity.
In addition, evidence shows that the impact of diet on gut micro-
biota may vary substantially between distinct species, strains and sex.
A comparative study between 7-week-old male Wistar and Sprague–
Dawley rats under HFD (45% energy from lipids and 17% from
sucrose) for 17 weeks suggested that the differences observed in the
gut microbial ecology may contribute to the worsened metabolic sce-
nario observed in the Wistar rat.60 In another study, 4 weeks of
methionine-restricted diet (0.15%) was able to promote significant
sex-specific changes in the intestinal microbiome of 20-week-old male
and female C57BL/6 mice, compared with mice on control diet con-
taining 0.65% methionine.274 Finally, even rodents from different pro-
duction facilities possess distinct gut microbiota, as reported in a
study using obesity/diabetes-prone C57Bl/6J and obesity/diabetes-
resistant 129S1/SvImJ mice from Jackson Laboratory and obesity-
prone/diabetes-resistant 129S6/SvEvTac mice from Taconic,
suggesting a significant contribution to differences in susceptibility to
DIO.262 Collectively, the available evidence emphasized the impor-
tance of careful choice of animals and diets when using models of
obesity and related metabolic conditions, particularly for studies
aimed at evaluating the effects on gut microbiota.
22
4 | CO NC LUSIO NS
Owing to the dearth of perfect animal models of obesity and
related metabolic diseases, selection of models that can be path-
ologically relevant for the scientific aims of a study is important.
Despite the underlying limitations and critical aspects, diet modi-
fication can be an appealing strategy, as unbalanced eating habit
is one of the main risk factors for the development of these
pathologies in humans. In addition, the slow and progressive evo-
lution of the disease and the continuum of associated pheno-
types (glucose intolerance, hyperglycaemia, insulin resistance,
hyperlipidaemia, glucotoxicity and lipotoxicity, oxidative stress and
inflammation and β-cell failure) better mimic human metabolic
diseases and allow evaluation of the potential of therapeutic or
nutraceutical interventions.
HFD and HSD, alone or in combination, are the most widely
used diets for inducing metabolic diseases associated with obesity
in rodent models. The percentage and type of each of the main
macromolecules (especially sugars and fats), the specific composition
of micronutrients, as well as the duration of exposure, are the key
factors determining the development of a desired model. In the case
of overt diabetes, the use of chemical inducers/accelerators of pan-
creatic β-cell damage (mainly STZ) should be additionally considered.
However, researchers should consider the innumerable variables
affecting the correct choice of an animal model of obesity and dis-
orders associated with obesity (MetS, prediabetes, T2DM and
NAFLD). In fact, besides diet type and composition, control diet and
protocol duration, the rodent species and strain used, as well as
their age and gender, can also significantly affect the results. In
addition, factors such as the origin of the animals and the way they
are housed and maintained can also bias the expected outcome.
Furthermore, as the main goal of animal models is to recapitulate
the various forms of human diseases, leveraging of
etic/epigenetic diversity to maximize a specific phenotype of inter-
est is clearly a compelling and powerful approach that warrants
further investigation.
All these conditions and variables demand careful evaluation,
such that the best model can be selected to achieve scientific results
with higher potential for translation into human disease-related
research. In addition to the use of efficient and overlapping methods,
definition of clear and universally accepted criteria for these diseases
in rodents, which can be reliably compared with those existing for dis-
ease diagnosis in humans, will be a key step towards improving pre-
clinical research and its ability to generate better tools for use in
humans.
CONF LICT OF IN TE RE ST
The authors declare no conflict of interest.
AUTHOR CONTRIBUTIONS
All the authors have substantially contributed to the final version,
which was revised and approved by all. I.P., S.N. and P.G. performed
the tables. A.A. and F.R. performed the first draft of figures.
PREGUIÇA ET AL.
FUNDING INF ORMATI ON
This work was also supported by European Regional Development
Fund (FEDER), through Programa Operacional Factores de Com-
petitividade COMPETE2020 (CENTRO-01-0145-FEDER-000012-
HealthyAging2020) and by National funds via Portuguese Science and
Technology Foundation (FCT): Strategic Projects UID/NEU/04539/
2013, UID/NEU/04539/2019, UIDB/04539/2020 and UIDP/04539/
2020 (CIBB); PhD Fellowship SFRH/BD/109017/2015; PTDC/SAU-
NUT/31712/2017, as well as by COMPETE-FEDER funds (POCI-01-
0145-FEDER-007440 and POCI-01-0145-FEDER-031712).
OR CID
Inês Preguiça https://orcid.org/0000-0002-7371-1079
André Alves https://orcid.org/0000-0002-8161-5239
Sara Nunes https://orcid.org/0000-0002-0240-9059
Rosa Fernandes https://orcid.org/0000-0001-7828-2296
Pedro Gomes https://orcid.org/0000-0003-1766-9133
Sofia D. Viana https://orcid.org/0000-0002-1316-1319
Flávio Reis https://orcid.org/0000-0003-3401-9554
RE FE RE NCE S
1. Ng M, Fleming T, Robinson M, et al. Global, regional, and national
prevalence of overweight and obesity in children and adults during
1980–2013: a systematic analysis for the Global Burden of Disease
Study 2013. Lancet. 2014;384(9945):766-781.
2. Pi-Sunyer X. The medical risks of obesity. Postgrad Med. 2009;
121(6):21-33.
3. Global BMIMC, Di Angelantonio E, Bhupathiraju Sh N, et al. Body-
mass index and all-cause mortality: individual-participant-data meta-
analysis of 239 prospective studies in four continents. Lancet. 2016;
388(10046):776-786.
4. Collaborators GBDO, Afshin A, Forouzanfar MH, et al. Health effects
of overweight and obesity in 195 countries over 25 years. N Engl J
Med. 2017;377(1):13-27.
gen- 5. Webb VL, Wadden TA. Intensive lifestyle intervention for obesity:
principles, practices, and results. Gastroenterology. 2017;152(7):
1752-1764.
6. Foright RM, Presby DM, Sherk VD, et al. Is regular exercise an effec-
tive strategy for weight loss maintenance? Physiol Behav. 2018;188:
86-93.
7. Teixeira-Lemos E, Nunes S, Teixeira F, Reis F. Regular physical exer-
cise training assists in preventing type 2 diabetes development:
focus on its antioxidant and anti-inflammatory properties. Cardiovasc
Diabetol. 2011;10:12.
8. Kim GW, Lin JE, Blomain ES, Waldman SA. Antiobesity pharmaco-
therapy: new drugs and emerging targets. Clin Pharmacol Ther. 2014;
95(1):53-66.
9. Bray GA, Fruhbeck G, Ryan DH, Wilding JP. Management of obesity.
Lancet. 2016;387(10031):1947-1956.
10. Kleinert M, Clemmensen C, Hofmann SM, et al. Animal models of
obesity and diabetes mellitus. Nat Rev Endocrinol. 2018;14(3):
140-162.
11. King AJ. The use of animal models in diabetes research. Br J
Pharmacol. 2012;166(3):877-894.
12. Preguica I, Alves A, Nunes S, et al. Diet-induced rodent models of
diabetic peripheral neuropathy, retinopathy and nephropathy. Nutri-
ents. 2020;12(1):250.
13. Bray GA, York DA, Fisler JS. Experimental obesity: a homeostatic
failure due to defective nutrient stimulation of the sympathetic ner-
vous system. Vitam Horm. 1989;45:1-125.
PREGUIÇA ET AL.
14. York DA. Lessons from animal models of obesity. Endocrinol Metab
Clin North Am. 1996;25(4):781-800.
15. van der Worp HB, Howells DW, Sena ES, et al. Can animal models
of disease reliably inform human studies? PLoS Med. 2010;7(3):
e1000245.
16. Beigrezaei S, Ghiasvand R, Feizi A, Iraj B. Relationship between die-
tary patterns and incidence of type 2 diabetes. Int J Prev Med. 2019;
10:122.
17. Rice Bradley BH. Dietary fat and risk for type 2 diabetes: a review of
recent research. Curr Nutr Rep. 2018;7(4):214-226.
18. Forouhi NG, Misra A, Mohan V, Taylor R, Yancy W. Dietary and
nutritional approaches for prevention and management of type
2 diabetes. BMJ. 2018;361:k2234.
19. Rodriguez-Monforte M, Sanchez E, Barrio F, Costa B, Flores-
Mateo G. Metabolic syndrome and dietary patterns: a systematic
review and meta-analysis of observational studies. Eur J Nutr. 2017;
56(3):925-947.
20. Huang D, Refaat M, Mohammedi K, Jayyousi A, Al Suwaidi J,
Abi KC. Macrovascular complications in patients with diabetes and
prediabetes. Biomed Res Int. 2017;2017:7839101.
21. Riazi K, Raman M, Taylor L, Swain MG, Shaheen AA. Dietary
patterns and components in nonalcoholic fatty liver disease
(NAFLD): what key messages can health care providers offer? Nutri-
ents. 2019;11(12).
22. Del Prato S. Role of glucotoxicity and lipotoxicity in the pathophysi-
ology of type 2 diabetes mellitus and emerging treatment strategies.
Diabet Med. 2009;26(12):1185-1192.
23. Akhtar DH, Iqbal U, Vazquez-Montesino LM, Dennis BB, Ahmed A.
Pathogenesis of insulin resistance and atherogenic dyslipidemia in
nonalcoholic fatty liver disease. J Clin Transl Hepatol. 2019;7(4):
362-370.
24. Bergman M. Pathophysiology of prediabetes and treatment implica-
tions for the prevention of type 2 diabetes mellitus. Endocrine. 2013;
43(3):504-513.
25. Savage DB, Petersen KF, Shulman GI. Disordered lipid metabolism
and the pathogenesis of insulin resistance. Physiol Rev. 2007;87(2):
507-520.
26. DeFronzo RA. Pathogenesis of type 2 diabetes mellitus. Med Clin
North Am. 2004;88(4):787-835. ix
27. Zheng Y, Ley SH, Hu FB. Global aetiology and epidemiology of type
2 diabetes mellitus and its complications. Nat Rev Endocrinol. 2018;
14(2):88-98.
28. Li Y, Xu W, Liao Z, et al. Induction of long-term glycemic control in
newly diagnosed type 2 diabetic patients is associated with improve-
ment of β-cell function. Diabetes Care. 2004;27(11):2597-2602.
29. Lebeaupin C, Vallee D, Hazari Y, Hetz C, Chevet E, Bailly-Maitre B.
Endoplasmic reticulum stress signalling and the pathogenesis of
non-alcoholic fatty liver disease. J Hepatol. 2018;69(4):927-947.
30. Quesada-Vázquez S, Aragonès G, Del Bas JM, Escoté X. Diet, Gut
Microbiota and Non-Alcoholic Fatty Liver Disease: Three Parts of
the Same Axis. Cells. 2020;9 (1):176. https://doi.org/10.3390/
cells9010176
31. Mohan S, Brown L, Ayyappan P. Endoplasmic reticulum stress: a
master regulator of metabolic syndrome. Eur J Pharmacol. 2019;860:
172553.
32. Lai M, Chandrasekera PC, Barnard ND. You are what you eat, or are
you? The challenges of translating high-fat-fed rodents to human
obesity and diabetes. Nutr Diabetes. 2014;4:e135.
33. Gasparin FRS, Carreno FO, Mewes JM, et al. Sex differences in
the development of hepatic steatosis in cafeteria diet-induced obe-
sity in young mice. Biochim Biophys Acta Mol Basis Dis. 2018;
1864(7):2495-2509.
34. Moore BJ. The cafeteria diet—an inappropriate tool for studies of
thermogenesis. J Nutr. 1987;117(2):227-231.
23
35. Speakman JR. Use of high-fat diets to study rodent obesity as a
model of human obesity. Int J Obes (Lond). 2019;43(8):1491-1492.
36. Aguila MB, Pinheiro Ada R, Parente LB, Mandarim-de-Lacerda CA.
Dietary effect of different high-fat diet on rat liver stereology. Liver
Int. 2003;23(5):363-370.
37. Flanagan AM, Brown JL, Santiago CA, Aad PY, Spicer LJ, Spicer MT.
High-fat diets promote insulin resistance through cytokine gene
expression in growing female rats. J Nutr Biochem. 2008;19(8):
505-513.
38. Taylor LE, Gillis EE, Musall JB, Baban B, Sullivan JC. High-fat diet-
induced hypertension is associated with a proinflammatory T cell
profile in male and female Dahl salt-sensitive rats. Am J Physiol Heart
Circ Physiol. 2018;315(6):H1713-H1723.
39. Bae CR, Hino J, Hosoda H, et al. Adipocyte-specific expression of C-
type natriuretic peptide suppresses lipid metabolism and adipocyte
hypertrophy in adipose tissues in mice fed high-fat diet. Sci Rep.
2018;8(1):2093.
40. Han L, Li T, Du M, Chang R, Zhan B, Mao X. Beneficial Effects of
Potentilla discolor Bunge Water Extract on Inflammatory Cytokines
Release and Gut Microbiota in High-Fat Diet and Streptozotocin-
Induced Type 2 Diabetic Mice. Nutrients. 2019;11 (3):670. https://
doi.org/10.3390/nu11030670
41. Buettner R, Parhofer KG, Woenckhaus M, et al. Defining high-fat-
diet rat models: metabolic and molecular effects of different fat
types. J Mol Endocrinol. 2006;36(3):485-501.
42. Do M, Lee E, Oh M-J, Kim Y, Park H-Y. High-Glucose or -Fructose
Diet Cause Changes of the Gut Microbiota and Metabolic Disorders
in Mice without Body Weight Change. Nutrients. 2018;10 (6):761.
https://doi.org/10.3390/nu10060761
43. Jurgens H, Haass W, Castaneda TR, et al. Consuming fructose-
sweetened beverages increases body adiposity in mice. Obes Res.
2005;13(7):1146-1156.
44. Hintze KJ, Benninghoff AD, Cho CE, Ward RE. Modeling the West-
ern diet for preclinical investigations. Adv Nutr. 2018;9(3):263-271.
45. Monsanto SP, Hintze KJ, Ward RE, Larson DP, Lefevre M,
Benninghoff AD. The new total Western diet for rodents does not
induce an overweight phenotype or alter parameters of metabolic
syndrome in mice. Nutr Res. 2016;36(9):1031-1044.
46. Bortolin RC, Vargas AR, Gasparotto J, et al. A new animal diet based
on human Western diet is a robust diet-induced obesity model: com-
parison to high-fat and cafeteria diets in term of metabolic and gut
microbiota disruption. Int J Obes (Lond). 2018;42(3):525-534.
47. Organization WH. Obesity and overweight. February 16, 2018;
www.who.int/news-room/fact-sheets/detail/obesity-and-
overweight
48. Bray GA, Kim KK, Wilding JPH, World OF. Obesity: a chronic
relapsing progressive disease process. A position statement of the
World Obesity Federation. Obes Rev. 2017;18(7):715-723.
49. Collaboration NCDRF. Worldwide trends in body-mass index,
underweight, overweight, and obesity from 1975 to 2016: a pooled
analysis of 2416 population-based measurement studies in 128.9
million children, adolescents, and adults. Lancet. 2017;390(10113):
2627-2642.
50. Despres JP. Body fat distribution and risk of cardiovascular disease:
an update. Circulation. 2012;126(10):1301-1313.
51. Neeland IJ, Turer AT, Ayers CR, et al. Dysfunctionlal adiposity and
the risk of prediabetes and type 2 diabetes in obese adults. JAMA.
2012;308(11):1150-1159.
52. Nuttall FQ. Body mass index: obesity, BMI, and health: a critical
review. Nutr Today. 2015;50(3):117-128.
53. Ross R, Neeland IJ, Yamashita S, et al. Waist circumference as a vital
sign in clinical practice: a Consensus Statement from the IAS and
ICCR Working Group on Visceral Obesity. Nat Rev Endocrinol. 2020;
16(3):177-189.
24
54. Wagner DR, Castaneda F, Bohman B, Sterr W. Comparison of a 2D
iPad application and 3D body scanner to air displacement plethys-
mography for measurement of body fat percentage. J Hum Nutr Diet.
2019;32(6):781-788.
55. Gibby JT, Njeru DK, Cvetko ST, Heiny EL, Creer AR, Gibby WA.
Whole-body computed tomography-based body mass and body fat
quantification: a comparison to hydrostatic weighing and air dis-
placement plethysmography. J Comput Assist Tomogr. 2017;41(2):
302-308.
56. Nickerson BS, Fedewa MV, Cicone Z, Esco MR. The relative accu-
racy of skinfolds compared to four-compartment estimates of body
composition. Clin Nutr. 2019;39(4):1112-1116.
57. Shen W, Wang Z, Punyanita M, et al. Adipose tissue quantification
by imaging methods: a proposed classification. Obes Res. 2003;11(1):
5-16.
58. Peig J, Green AJ. The paradigm of body condition: a critical
reappraisal of current methods based on mass and length. Functional
Ecology. 2010;24(6):1323-1332.
59. Lutz TA, Woods SC. Overview of animal models of obesity. Curr
Protoc Pharmacol. 2012;58(1):5-61. Chapter 5:Unit5 61
60. Marques C, Meireles M, Norberto S, et al. High-fat diet-induced
obesity rat model: a comparison between Wistar and Sprague–
Dawley Rat. Adipocyte. 2016;5(1):11-21.
61. Cheng HS, Ton SH, Phang SCW, Tan JBL, Abdul KK. Increased
susceptibility of post-weaning rats on high-fat diet to metabolic
syndrome. J Adv Res. 2017;8(6):743-752.
62. Alexander J, Chang GQ, Dourmashkin JT, Leibowitz SF. Distinct
phenotypes of obesity-prone AKR/J, DBA2J and C57BL/6J mice
compared to control strains. Int J Obes (Lond). 2006;30(1):50-59.
63. Lee YS, Li P, Huh JY, et al. Inflammation is necessary for long-term
but not short-term high-fat diet-induced insulin resistance. Diabetes.
2011;60(10):2474-2483.
64. Turner N, Kowalski GM, Leslie SJ, et al. Distinct patterns of tissue-
specific lipid accumulation during the induction of insulin resistance
in mice by high-fat feeding. Diabetologia. 2013;56(7):1638-1648.
65. Montgomery MK, Hallahan NL, Brown SH, et al. Mouse strain-
dependent variation in obesity and glucose homeostasis in response
to high-fat feeding. Diabetologia. 2013;56(5):1129-1139.
66. Martin-Cordero L, Galvez I, Hinchado MD, Ortega E. β2 adrenergic
regulation of the phagocytic and microbicide capacity of macro-
phages from obese and lean mice: effects of exercise. Nutrients.
2019;11(11):2721.
67. Galvez I, Martin-Cordero L, Hinchado MD, Alvarez-Barrientos A,
Ortega E. Anti-inflammatory effect of β2 adrenergic stimulation on
circulating monocytes with a pro-inflammatory state in high-fat diet-
induced obesity. Brain Behav Immun. 2019;80:564-572.
68. Levin BE, Dunn-Meynell AA, Balkan B, Keesey RE. Selective breed-
ing for diet-induced obesity and resistance in Sprague–Dawley rats.
Am J Physiol. 1997;273(2 Pt 2):R725-R730.
69. Souza GF, Solon C, Nascimento LF, et al. Defective regulation of
POMC precedes hypothalamic inflammation in diet-induced obesity.
Sci Rep. 2016;6:29290.
70. Chang S, Graham B, Yakubu F, Lin D, Peters JC, Hill JO. Metabolic
differences between obesity-prone and obesity-resistant rats.
Am J Physiol. 1990;259(6 Pt 2):R1103-R1110.
71. MacLean PS, Higgins JA, Johnson GC, et al. Enhanced metabolic effi-
ciency contributes to weight regain after weight loss in obesity-
prone rats. Am J Physiol Regul Integr Comp Physiol. 2004;287(6):
R1306-R1315.
72. Aziz AA, Kenney LS, Goulet B, Abdel-Aal el S. Dietary starch type
affects body weight and glycemic control in freely fed but not
energy-restricted obese rats. J Nutr. 2009;139(10):1881-1889.
73. Belobrajdic DP, King RA, Christophersen CT, Bird AR. Dietary resis-
tant starch dose-dependently reduces adiposity in obesity-prone
and obesity-resistant male rats. Nutr Metab (Lond). 2012;9(1):93.
PREGUIÇA ET AL.
74. Lozano I, Van der Werf R, Bietiger W, et al. High-fructose and high-
fat diet-induced disorders in rats: impact on diabetes risk, hepatic
and vascular complications. Nutr Metab (Lond). 2016;13:15.
75. Hafizur RM, Raza SA, Chishti S, Shaukat S, Ahmed A. A ‘humanized’
rat model of pre-diabetes by high fat diet-feeding to weaning wistar
rats. Integr Obes Diabetes. 2015;1(2):44-48.
76. Sugano M, Yamato H, Hayashi T, et al. High-fat diet in low-dose-
streptozotocin-treated heminephrectomized rats induces all features
of human type 2 diabetic nephropathy: a new rat model of diabetic
nephropathy. Nutr Metab Cardiovasc Dis. 2006;16(7):477-484.
77. Oliveira LS, Santos DA, Barbosa-da-Silva S, Mandarim-de-
Lacerda CA, Aguila MB. The inflammatory profile and liver damage
of a sucrose-rich diet in mice. J Nutr Biochem. 2014;25(2):193-200.
78. Roopchand DE, Carmody RN, Kuhn P, et al. Dietary polyphenols
promote growth of the gut bacterium Akkermansia muciniphila and
attenuate high-fat diet-induced metabolic syndrome. Diabetes.
2015;64(8):2847-2858.
79. Meng Q, Lai YC, Kelly NJ, et al. Development of a mouse model of
metabolic syndrome, pulmonary hypertension, and heart failure with
preserved ejection fraction. Am J Respir Cell Mol Biol. 2017;56(4):
497-505.
80. Malik VS, Popkin BM, Bray GA, Despres JP, Hu FB. Sugar-
sweetened beverages, obesity, type 2 diabetes mellitus, and cardio-
vascular disease risk. Circulation. 2010;121(11):1356-1364.
81. Tappy L, Le KA. Metabolic effects of fructose and the worldwide
increase in obesity. Physiol Rev. 2010;90(1):23-46.
82. Bocarsly ME, Powell ES, Avena NM, Hoebel BG. High-fructose corn
syrup causes characteristics of obesity in rats: increased body
weight, body fat and triglyceride levels. Pharmacol Biochem Behav.
2010;97(1):101-106.
83. Tillman EJ, Morgan DA, Rahmouni K, Swoap SJ. Three months of
high-fructose feeding fails to induce excessive weight gain or leptin
resistance in mice. PLoS ONE. 2014;9(9):e107206.
84. Sumiyoshi M, Sakanaka M, Kimura Y. Chronic intake of high-fat and
high-sucrose diets differentially affects glucose intolerance in mice.
J Nutr. 2006;136(3):582-587.
85. Yang ZH, Miyahara H, Takeo J, Katayama M. Diet high in fat and
sucrose induces rapid onset of obesity-related metabolic syndrome
partly through rapid response of genes involved in lipogenesis,
insulin signalling and inflammation in mice. Diabetol Metab Syndr.
2012;4(1):32.
86. Zhang L, Song H, Ge Y, Ji G, Yao Z. Temporal relationship between
diet-induced steatosis and onset of insulin/leptin resistance in male
Wistar rats. PLoS ONE. 2015;10(2):e0117008.
87. Roberts CK, Hevener AL, Barnard RJ. Metabolic syndrome and
insulin resistance: underlying causes and modification by exercise
training. Compr Physiol. 2013;3(1):1-58.
88. Kassi E, Pervanidou P, Kaltsas G, Chrousos G. Metabolic syndrome:
definitions and controversies. BMC Med. 2011;9:48.
89. Alberti KG, Eckel RH, Grundy SM, et al. Harmonizing the metabolic
syndrome: a joint interim statement of the International Diabetes
Federation Task Force on Epidemiology and Prevention; National
Heart, Lung, and Blood Institute; American Heart Association; World
Heart Federation; International Atherosclerosis Society; and Interna-
tional Association for the Study of Obesity. Circulation. 2009;
120(16):1640-1645.
90. Nolan PB, Carrick-Ranson G, Stinear JW, Reading SA, Dalleck LC.
Prevalence of metabolic syndrome and metabolic syndrome compo-
nents in young adults: a pooled analysis. Prev Med Rep. 2017;7:
211-215.
91. Saklayen MG. The global epidemic of the metabolic syndrome. Curr
Hypertens Rep. 2018;20(2):12.
92. Gurka MJ, Filipp SL, DeBoer MD. Geographical variation in the
prevalence of obesity, metabolic syndrome, and diabetes among US
adults. Nutr Diabetes. 2018;8(1):14.
PREGUIÇA ET AL.
93. Sigit FS, Tahapary DL, Trompet S, et al. The prevalence of metabolic
syndrome and its association with body fat distribution in middle-
aged individuals from Indonesia and the Netherlands: a cross-
sectional analysis of two population-based studies. Diabetol Metab
Syndr. 2020;12:2.
94. Lind L. Genome-wide association study of the metabolic syndrome
in UK biobank. Metab Syndr Relat Disord. 2019;17(10):505-511.
95. Prasad G, Bandesh K, Giri AK, et al. Genome-wide association study
of metabolic syndrome reveals primary genetic variants at CETP
locus in Indians. Biomolecules. 2019;9(8):321.
96. Wei ZY, Liu JJ, Zhan XM, Feng HM, Zhang YY. Dietary patterns and
the risk of metabolic syndrome in Chinese adults: a population-
based cross-sectional study. Public Health Nutr. 2018;21(13):2409-
2416.
97. Wang LX, Gurka MJ, Deboer MD. Metabolic syndrome severity and
lifestyle factors among adolescents. Minerva Pediatr. 2018;70(5):
467-475.
98. Di Daniele N, Noce A, Vidiri MF, et al. Impact of Mediterranean diet
on metabolic syndrome, cancer and longevity. Oncotarget. 2017;
8(5):8947-8979.
99. Wong SK, Chin KY, Suhaimi FH, Fairus A, Ima-Nirwana S. Animal
models of metabolic syndrome: a review. Nutr Metab (Lond). 2016;
13:65.
100. Wanrooy BJ, Kumar KP, Wen SW, Qin CX, Ritchie RH, Wong CHY.
Distinct contributions of hyperglycemia and high-fat feeding in met-
abolic syndrome-induced neuroinflammation. J Neuroinflammation.
2018;15(1):293.
101. Mohammad Saifur Rohman ML, Nugroho DA, Nashi W,
Nugraheini NIP, Sardjono TW. Development of an experimental
model of metabolic syndrome in Sprague Dawley rat. Res J Life Sci.
2017;4:76-86.
102. Heydemann A. An overview of murine high fat diet as a model for
type 2 diabetes mellitus. J Diabetes Res. 2016;2016:2902351.
103. Hu S, Wang L, Yang D, et al. Dietary fat, but not protein or carbohy-
drate, regulates energy intake and causes adiposity in mice. Cell
Metab. 2018;28(3):415-431. e414
104. Ghezzi AC, Cambri LT, Botezelli JD, Ribeiro C, Dalia RA, de
Mello MA. Metabolic syndrome markers in wistar rats of different
ages. Diabetol Metab Syndr. 2012;4(1):16.
105. Nunes-Souza V, Cesar-Gomes CJ, Da Fonseca LJ, Guedes Gda S,
Smaniotto S, Rabelo LA. Aging increases susceptibility to high fat
diet-induced metabolic syndrome in C57BL/6 mice: improvement in
glycemic and lipid profile after antioxidant therapy. Oxid Med Cell
Longev. 2016;2016:1987960.
106. Lasker S, Rahman MM, Parvez F, et al. High-fat diet-induced meta-
bolic syndrome and oxidative stress in obese rats are ameliorated by
yogurt supplementation. Sci Rep. 2019;9(1):20026.
107. Oron-Herman M, Kamari Y, Grossman E, et al. Metabolic syndrome:
comparison of the two commonly used animal models.
Am J Hypertens. 2008;21(9):1018-1022.
108. Ritze Y, Bardos G, D'Haese JG, et al. Effect of high sugar intake on
glucose transporter and weight regulating hormones in mice and
humans. PLoS ONE. 2014;9(7):e101702.
109. Montgomery MK, Fiveash CE, Braude JP, et al. Disparate metabolic
response to fructose feeding between different mouse strains. Sci
Rep. 2015;5:18474.
110. Aslam M, Madhu SV. Development of metabolic syndrome in high-
sucrose diet fed rats is not associated with decrease in adiponectin
levels. Endocrine. 2017;58(1):59-65.
111. Aguilera-Mendez A, Hernandez-Equihua MG, Rueda-Rocha AC, et al.
Protective effect of supplementation with biotin against high-fruc-
tose-induced metabolic syndrome in rats. Nutr Res. 2018;57:86-96.
112. Nunes S, Soares E, Fernandes J, et al. Early cardiac changes in a rat
model of prediabetes: brain natriuretic peptide overexpression
seems to be the best marker. Cardiovasc Diabetol. 2013;12:44.
25
113. Moreno-Fernandez S, Garces-Rimon M, Vera G, Astier J,
Landrier JF, Miguel M. High fat/high glucose diet induces metabolic
syndrome in an experimental rat model. Nutrients. 2018;10(10):
1502.
114. Masi LN, Martins AR, Crisma AR, et al. Combination of a high-fat
diet with sweetened condensed milk exacerbates inflammation and
insulin resistance induced by each separately in mice. Sci Rep. 2017;
7(1):3937.
115. Hostalek U. Global epidemiology of prediabetes—present and future
perspectives. Clin Diabetes Endocrinol. 2019;5:5.
116. Tabak AG, Herder C, Rathmann W, Brunner EJ, Kivimaki M. Predia-
betes: a high-risk state for diabetes development. Lancet. 2012;
379(9833):2279-2290.
117. Wilson ML. Prediabetes: beyond the borderline. Nurs Clin North Am.
2017;52(4):665-677.
118. Faerch K, Borch-Johnsen K, Holst JJ, Vaag A. Pathophysiology and
aetiology of impaired fasting glycaemia and impaired glucose toler-
ance: does it matter for prevention and treatment of type 2 diabe-
tes? Diabetologia. 2009;52(9):1714-1723.
119. American Diabetes A. 2. Classification and diagnosis of diabetes:
standards of medical care in diabetes-2019. Diabetes Care. 2019;
42(Suppl 1):S13-S28.
120. Ausk KJ, Boyko EJ, Ioannou GN. Insulin resistance predicts mortality
in nondiabetic individuals in the U.S. Diabetes Care. 2010;33(6):
1179-1185.
121. Hedblad B, Nilsson P, Engstrom G, Berglund G, Janzon L. Insulin
resistance in non-diabetic subjects is associated with increased inci-
dence of myocardial infarction and death. Diabet Med. 2002;19(6):
470-475.
122. Abdul-Ghani MA, DeFronzo RA. Pathophysiology of prediabetes.
Curr Diab Rep. 2009;9(3):193-199.
123. Saravanan N, Patil MA, Kumar PU, Suryanarayana P, Reddy GB. Die-
tary ginger improves glucose dysregulation in a long-term high-fat
high-fructose fed prediabetic rat model. Indian J Exp Biol. 2017;
55(3):142-150.
124. Tanajak P, Sa-Nguanmoo P, Apaijai N, et al. Comparisons of cardi-
oprotective efficacy between fibroblast growth factor 21 and
dipeptidyl peptidase-4 inhibitor in prediabetic rats. Cardiovasc Ther.
2017;35(4):e12263.
125. Sweazea KL, Lekic M, Walker BR. Comparison of mechanisms
involved in impaired vascular reactivity between high sucrose and
high fat diets in rats. Nutr Metab (Lond). 2010;7:48.
126. Mosser RE, Maulis MF, Moulle VS, et al. High-fat diet-induced β-cell
proliferation occurs prior to insulin resistance in C57Bl/6J male
mice. Am J Physiol Endocrinol Metab. 2015;308(7):E573-E582.
127. Small L, Brandon AE, Turner N, Cooney GJ. Modeling insulin resis-
tance in rodents by alterations in diet: what have high-fat and high-
calorie diets revealed? Am J Physiol Endocrinol Metab. 2018;314(3):
E251-E265.
128. Yang T, Xu WJ, York H, Liang NC. Diet choice patterns in rodents
depend on novelty of the diet, exercise, species, and sex. Physiol
Behav. 2017;176:149-158.
129. Catta-Preta M, Martins MA, Cunha Brunini TM, Mendes-
Ribeiro AC, Mandarim-de-Lacerda CA, Aguila MB. Modulation
of cytokines, resistin, and distribution of adipose tissue in
C57BL/6 mice by different high-fat diets. Nutrition. 2012;28(2):
212-219.
130. Abdel-Hamid AAM, Firgany AEL. Correlation between pancreatic
mast cells and the low grade inflammation in adipose tissue of
experimental prediabetes. Acta Histochem. 2019;121(1):35-42.
131. Winzell MS, Ahren B. The high-fat diet-fed mouse: a model for
studying mechanisms and treatment of impaired glucose tolerance
and type 2 diabetes. Diabetes. 2004;53(Suppl 3):S215-S219.
132. Chen GC, Huang CY, Chang MY, et al. Two unhealthy dietary habits
featuring a high fat content and a sucrose-containing beverage
26
intake, alone or in combination, on inducing metabolic syndrome in
Wistar rats and C57BL/6J mice. Metabolism. 2011;60(2):155-164.
133. Thresher JS, Podolin DA, Wei Y, Mazzeo RS, Pagliassotti MJ. Com-
parison of the effects of sucrose and fructose on insulin action and
glucose tolerance. Am J Physiol Regul Integr Comp Physiol. 2000;
279(4):R1334-R1340.
134. Togo J, Hu S, Li M, Niu C, Speakman JR. Impact of dietary sucrose
on adiposity and glucose homeostasis in C57BL/6J mice depends on
mode of ingestion: liquid or solid. Mol Metab. 2019;27:22-32.
135. Surwit RS, Feinglos MN, Rodin J, et al. Differential effects of fat and
sucrose on the development of obesity and diabetes in C57BL/6J
and A/J mice. Metabolism. 1995;44(5):645-651.
136. Burgeiro A, Cerqueira MG, Varela-Rodriguez BM, et al. Glucose and
lipid dysmetabolism in a rat model of prediabetes induced by a high-
sucrose diet. Nutrients. 2017;9(6):638.
137. Kanazawa M, Xue CY, Kageyama H, et al. Effects of a high-sucrose
diet on body weight, plasma triglycerides, and stress tolerance. Nutr
Rev. 2003;61(5 Pt 2):S27-S33.
138. Alves MRP, Boia R, Campos EJ, et al. Subtle thinning of retinal layers
without overt vascular and inflammatory alterations in a rat model
of prediabetes. Mol Vis. 2018;24:353-366.
139. Soares E, Prediger RD, Nunes S, et al. Spatial memory impairments
in a prediabetic rat model. Neuroscience. 2013;250:565-577.
140. Pham N, Dhar A, Khalaj S, Desai K, Taghibiglou C. Down regulation
of brain cellular prion protein in an animal model of insulin resis-
tance: possible implication in increased prevalence of stroke in pre-
diabetics/diabetics. Biochem Biophys Res Commun. 2014;448(2):
151-156.
141. Alzamendi A, Giovambattista A, Raschia A, et al. Fructose-rich diet-
induced abdominal adipose tissue endocrine dysfunction in normal
male rats. Endocrine. 2009;35(2):227-232.
142. Tappy L, Le KA, Tran C, Paquot N. Fructose and metabolic diseases:
new findings, new questions. Nutrition. 2010;26(11–12):1044-1049.
143. Tran LT, Yuen VG, McNeill JH. The fructose-fed rat: a review on the
mechanisms of fructose-induced insulin resistance and hyperten-
sion. Mol Cell Biochem. 2009;332(1–2):145-159.
144. Miranda CA, Schonholzer TE, Kloppel E, et al. Repercussions of low
fructose-drinking water in male rats. An Acad Bras Cienc. 2019;91(1):
e20170705.
145. Francini F, Massa ML, Polo MP, Villagarcia H, Castro MC,
Gagliardino JJ. Control of liver glucokinase activity: a potential new
target for incretin hormones? Peptides. 2015;74:57-63.
146. Kowalski GM, Kraakman MJ, Mason SA, Murphy AJ, Bruce CR. Res-
olution of glucose intolerance in long-term high-fat, high-sucrose-
fed mice. J Endocrinol. 2017;233(3):269-279.
147. Gamede M, Mabuza L, Ngubane P, Khathi A. The effects of plant-
derived oleanolic acid on selected parameters of glucose homeosta-
sis in a diet-induced pre-diabetic rat model. Molecules. 2018;
23(4):794.
148. Panchal SK, Brown L. Rodent Models for Metabolic Syndrome
Research. Journal of Biomedicine and Biotechnology. 2011;2011
1–14. https://doi.org/10.1155/2011/351982
149. Lomba A, Milagro FI, Garcia-Diaz DF, Marti A, Campion J,
Martinez JA. Obesity induced by a pair-fed high fat sucrose diet:
methylation and expression pattern of genes related to energy
homeostasis. Lipids Health Dis. 2010;9:60.
150. Chun MR, Lee YJ, Kim KH, et al. Differential effects of high-
carbohydrate and high-fat diet composition on muscle insulin resis-
tance in rats. J Korean Med Sci. 2010;25(7):1053-1059.
151. Sato A, Kawano H, Notsu T, et al. Antiobesity effect of
eicosapentaenoic acid in high-fat/high-sucrose diet-induced obesity:
importance of hepatic lipogenesis. Diabetes. 2010;59(10):2495-
2504.
PREGUIÇA ET AL.
152. van den Brom CE, Boly CA, Bulte CSE, van den Akker RFP,
Kwekkeboom RFJ, Loer SA, Boer C, Bouwman RA. Myocardial Per-
fusion and Function Are Distinctly Altered by Sevoflurane Anesthe-
sia in Diet-Induced Prediabetic Rats. Journal of Diabetes Research.
2016;2016:1–9. https://doi.org/10.1155/2016/5205631
153. Vatandoust N, Rami F, Salehi AR, et al. Novel high-fat diet formula-
tion and streptozotocin treatment for induction of prediabetes and
type 2 diabetes in rats. Adv Biomed Res. 2018;7:107.
154. Saeedi P, Petersohn I, Salpea P, Malanda B, Karuranga S, Unwin N,
Colagiuri S, Guariguata L, Motala AA, Ogurtsova K, Shaw JE,
Bright D, Williams R. Global and regional diabetes prevalence esti-
mates for 2019 and projections for 2030 and 2045: Results from
the International Diabetes Federation Diabetes Atlas, 9th edition.
Diabetes Research and Clinical Practice. 2019;157:107843. https://
doi.org/10.1016/j.diabres.2019.107843
155. Bowe JE, Franklin ZJ, Hauge-Evans AC, King AJ, Persaud SJ,
Jones PM. Metabolic phenotyping guidelines: assessing glucose
homeostasis in rodent models. J Endocrinol. 2014;222(3):G13-G25.
156. Andrikopoulos S, Blair AR, Deluca N, Fam BC, Proietto J. Evaluating
the glucose tolerance test in mice. Am J Physiol Endocrinol Metab.
2008;295(6):E1323-E1332.
157. Heikkinen S, Argmann CA, Champy MF, Auwerx J. Evaluation of
glucose homeostasis. Curr Protoc Mol Biol. 2007;77(1): Chapter 29:
Unit 29B.3.
158. Wang Z, Yang Y, Xiang X, Zhu Y, Men J, He M. Estimation of the
normal range of blood glucose in rats. Wei Sheng Yan Jiu. 2010;
39(2):133-137. 142
159. Vander Werf R, Walter C, Bietiger W, et al. Beneficial effects of
cherry consumption as a dietary intervention for metabolic, hepatic
and vascular complications in type 2 diabetic rats. Cardiovasc
Diabetol. 2018;17(1):104.
160. Clee SM, Attie AD. The genetic landscape of type 2 diabetes in mice.
Endocr Rev. 2007;28(1):48-83.
161. Leiter EH. Selecting the “right” mouse model for metabolic syn-
drome and type 2 diabetes research. Methods Mol Biol. 2009;560:
1-17.
162. Benede-Ubieto R, Estevez-Vazquez O, Ramadori P, Cubero FJ,
Nevzorova YA. Guidelines and considerations for metabolic
tolerance tests in mice. Diabetes Metab Syndr Obes. 2020;13:
439-450.
163. Kale VP, Joshi GS, Gohil PB, Jain MR. Effect of fasting duration on
clinical pathology results in Wistar rats. Vet Clin Pathol. 2009;38(3):
361-366.
164. Srinivasan K, Ramarao P. Animal models in type 2 diabetes research:
an overview. Indian J Med Res. 2007;125(3):451-472.
165. Chatzigeorgiou A, Halapas A, Kalafatakis K, Kamper E. The use of
animal models in the study of diabetes mellitus. In Vivo. 2009;23(2):
245-258.
166. Kuwabara WMT, Panveloski-Costa AC, Yokota CNF, et al. Compari-
son of Goto–Kakizaki rats and high fat diet-induced obese rats: are
they reliable models to study type 2 diabetes mellitus? PLoS ONE.
2017;12(12):e0189622.
167. Stark AH, Timar B, Madar Z. Adaptation of Sprague Dawley rats to
long-term feeding of high fat or high fructose diets. Eur J Nutr.
2000;39(5):229-234.
168. Patel J, Iyer A, Brown L. Evaluation of the chronic complications of
diabetes in a high fructose diet in rats. Indian J Biochem Biophys.
2009;46(1):66-72.
169. Rohlmann A, Basli K, Bergmann G. Analysis of the tension of the
elbow joint before and after resection of the radius head. Biomed
Tech (Berl). 1986;31(10):230-239.
170. Matheus VA, Monteiro L, Oliveira RB, Maschio DA, Collares-
Buzato CB. Butyrate reduces high-fat diet-induced metabolic
PREGUIÇA ET AL.
alterations, hepatic steatosis and pancreatic beta cell and intestinal
barrier dysfunctions in prediabetic mice. Exp Biol Med (Maywood).
2017;242(12):1214-1226.
171. Surwit RS, Kuhn CM, Cochrane C, McCubbin JA, Feinglos MN. Diet-
induced type II diabetes in C57BL/6J mice. Diabetes. 1988;37(9):
1163-1167.
172. Guo A, Daniels NA, Thuma J, McCall KD, Malgor R, Schwartz FL.
Diet is critical for prolonged glycemic control after short-term insulin
treatment in high-fat diet-induced type 2 diabetic male mice. PLoS
ONE. 2015;10(1):e0117556.
173. Kleindienst A, Battault S, Belaidi E, et al. Exercise does not activate
the β3 adrenergic receptor-eNOS pathway, but reduces inducible
NOS expression to protect the heart of obese diabetic mice. Basic
Res Cardiol. 2016;111(4):40.
174. Winters WD, Huo YS, Yao DL. Inhibition of the progression of type
2 diabetes in the C57BL/6J mouse model by an anti-diabetes herbal
formula. Phytother Res. 2003;17(6):591-598.
175. Chalkley SM, Hettiarachchi M, Chisholm DJ, Kraegen EW. Long-
term high-fat feeding leads to severe insulin resistance but not dia-
betes in Wistar rats. Am J Physiol Endocrinol Metab. 2002;282(6):
E1231-E1238.
176. Peterson R, Shaw WN, Neel MA, Little LA, Eichberg J. Zucker dia-
betic fatty rat as a model for non-insulin-dependent diabetes
mellitus. ILAR News. 1990;32:16-19.
177. Corsetti JP, Sparks JD, Peterson RG, Smith RL, Sparks CE. Effect of
dietary fat on the development of non-insulin dependent diabetes
mellitus in obese Zucker diabetic fatty male and female rats. Athero-
sclerosis. 2000;148(2):231-241.
178. Ferreira L, Teixeira-de-Lemos E, Pinto F, Parada B, Mega C,
Vala H, Pinto R, Garrido P, Sereno J, Fernandes R, Santos P,
Velada I, Melo A, Nunes S, Teixeira F, Reis F. Effects of Sitagliptin
Treatment on Dysmetabolism, Inflammation, and Oxidative Stress
in an Animal Model of Type 2 Diabetes (ZDF Rat). Mediators of
Inflammation. 2010;2010:1–11. https://doi.org/10.1155/2010/
592760
179. Uozumi K, Ohno N, Ishizuka K, Iwahashi M, Hanada S, Arima T.
Adult T-cell leukaemia in patients with OKT4 epitope deficiency. Br
J Haematol. 1991;79(4):651-652.
180. Kalaki-Jouybari F, Shanaki M, Delfan M, Gorgani-Firouzjae S,
Khakdan S. High-intensity interval training (HIIT) alleviated NAFLD
feature via miR-122 induction in liver of high-fat high-fructose diet
induced diabetic rats. Arch Physiol Biochem. 2018;1-8.
181. Balakumar M, Raji L, Prabhu D, et al. High-fructose diet is as
detrimental as high-fat diet in the induction of insulin resistance and
diabetes mediated by hepatic/pancreatic endoplasmic reticulum
(ER) stress. Mol Cell Biochem. 2016;423(1–2):93-104.
182. Skovso S. Modeling type 2 diabetes in rats using high fat diet and
streptozotocin. J Diabetes Investig. 2014;5(4):349-358.
183. Mansor LS, Gonzalez ER, Cole MA, et al. Cardiac metabolism in a
new rat model of type 2 diabetes using high-fat diet with low dose
streptozotocin. Cardiovasc Diabetol. 2013;12:136.
184. Guo XX, Wang Y, Wang K, Ji BP, Zhou F. Stability of a type
2 diabetes rat model induced by high-fat diet feeding with low-dose
streptozotocin injection. J Zhejiang Univ Sci B. 2018;19(7):559-569.
185. Chen M, Xiao D, Liu W, et al. Intake of Ganoderma lucidum polysac-
charides reverses the disturbed gut microbiota and metabolism in
type 2 diabetic rats. Int J Biol Macromol. 2019;155:890-902.
186. Lee JY, Jeong EA, Kim KE, et al. TonEBP/NFAT5 haploinsufficiency
attenuates hippocampal inflammation in high-fat diet/
streptozotocin-induced diabetic mice. Sci Rep. 2017;7(1):7837.
187. Leguina-Ruzzi A, Ortiz R, Velarde V. The streptozotocin-high fat diet
induced diabetic mouse model exhibits severe skin damage and
alterations in local lipid mediators. Biom J. 2018;41(5):328-332.
188. Goodarzi G, Shirgir A, Alavi S, Khoshi A. Effect of insulin-glucose
metabolism compared with obesity on adipose omentin gene
27
expression in different models of diabetic C57BL/6 mice. Diabetol
Metab Syndr. 2019;11:65.
189. Fang X, Xia T, Xu F, Wu H, Ma Z, Zhao X, Gu X. Isoflurane aggra-
vates peripheral and central insulin resistance in high-fat diet/-
streptozocin-induced type 2 diabetic mice. Brain Research. 2020;
1727:146511. https://doi.org/10.1016/j.brainres.2019.146511
190. Srinivasan K, Viswanad B, Asrat L, Kaul CL, Ramarao P. Combination
of high-fat diet-fed and low-dose streptozotocin-treated rat: a
model for type 2 diabetes and pharmacological screening. Pharmacol
Res. 2005;52(4):313-320.
191. Barriere DA, Noll C, Roussy G, et al. Combination of high-fat/high-
fructose diet and low-dose streptozotocin to model long-term type-
2 diabetes complications. Sci Rep. 2018;8(1):424.
192. Obrosov A, Shevalye H, Coppey LJ, Yorek MA. Effect of tempol on
peripheral neuropathy in diet-induced obese and high-fat fed/low-
dose streptozotocin-treated C57Bl6/J mice. Free Radic Res. 2017;
51(4):360-367.
193. Younossi ZM, Koenig AB, Abdelatif D, Fazel Y, Henry L, Wymer M.
Global epidemiology of nonalcoholic fatty liver disease-Meta-
analytic assessment of prevalence, incidence, and outcomes.
Hepatology. 2016;64(1):73-84.
194. Cohen JC, Horton JD, Hobbs HH. Human fatty liver disease: old
questions and new insights. Science. 2011;332(6037):1519-1523.
195. Nakamura A, Terauchi Y. Lessons from mouse models of high-fat
diet-induced NAFLD. Int J Mol Sci. 2013;14(11):21240-21257.
196. Ito M, Suzuki J, Tsujioka S, et al. Longitudinal analysis of murine
steatohepatitis model induced by chronic exposure to high-fat diet.
Hepatol Res. 2007;37(1):50-57.
197. Gauthier MS, Favier R, Lavoie JM. Time course of the development
of non-alcoholic hepatic steatosis in response to high-fat diet-
induced obesity in rats. Br J Nutr. 2006;95(2):273-281.
198. Omagari K, Kato S, Tsuneyama K, et al. Effects of a long-term high-
fat diet and switching from a high-fat to low-fat, standard diet on
hepatic fat accumulation in Sprague–Dawley rats. Dig Dis Sci. 2008;
53(12):3206-3212.
199. McCuskey RS, Ito Y, Robertson GR, McCuskey MK, Perry M,
Farrell GC. Hepatic microvascular dysfunction during evolution of
dietary steatohepatitis in mice. Hepatology. 2004;40(2):386-393.
200. Machado MV, Michelotti GA, Xie G, et al. Correction: Mouse models
of diet-induced nonalcoholic steatohepatitis reproduce the hetero-
geneity of the human disease. PLoS ONE. 2015;10(6):e0132315.
201. Wolf MJ, Adili A, Piotrowitz K, et al. Metabolic activation of
intrahepatic CD8+ T cells and NKT cells causes nonalcoholic
steatohepatitis and liver cancer via cross-talk with hepatocytes. Can-
cer Cell. 2014;26(4):549-564.
202. Miura K, Kodama Y, Inokuchi S, et al. Toll-like receptor 9 promotes
steatohepatitis by induction of interleukin-1β in mice. Gastroenterol-
ogy. 2010;139(1):323-334. e327
203. Uto H, Nakanishi C, Ido A, et al. The peroxisome proliferator-
activated receptor-gamma agonist, pioglitazone, inhibits fat accumu-
lation and fibrosis in the livers of rats fed a choline-deficient, L-
amino acid-defined diet. Hepatol Res. 2005;32(4):235-242.
204. Kamada Y, Matsumoto H, Tamura S, et al. Hypoadiponectinemia
accelerates hepatic tumor formation in a nonalcoholic
steatohepatitis mouse model. J Hepatol. 2007;47(4):556-564.
205. Matsumoto M, Hada N, Sakamaki Y, et al. An improved mouse
model that rapidly develops fibrosis in non-alcoholic steatohepatitis.
Int J Exp Pathol. 2013;94(2):93-103.
206. Matsuzawa N, Takamura T, Kurita S, et al. Lipid-induced oxidative
stress causes steatohepatitis in mice fed an atherogenic diet.
Hepatology. 2007;46(5):1392-1403.
207. Charlton M, Krishnan A, Viker K, et al. Fast food diet mouse: novel
small animal model of NASH with ballooning, progressive fibrosis,
and high physiological fidelity to the human condition. Am J Physiol
Gastrointest Liver Physiol. 2011;301(5):G825-G834.
28
208. Mamikutty N, Thent ZC, Haji SF. Fructose-drinking water induced
nonalcoholic fatty liver disease and ultrastructural alteration of
hepatocyte mitochondria in male wistar rat. Biomed Res Int. 2015;
2015:895961.
209. Spruss A, Kanuri G, Wagnerberger S, Haub S, Bischoff SC,
Bergheim I. Toll-like receptor 4 is involved in the development of
fructose-induced hepatic steatosis in mice. Hepatology. 2009;50(4):
1094-1104.
210. Asgharpour A, Cazanave SC, Pacana T, et al. A diet-induced animal
model of non-alcoholic fatty liver disease and hepatocellular cancer.
J Hepatol. 2016;65(3):579-588.
211. Nishikawa S, Yasoshima A, Doi K, Nakayama H, Uetsuka K. Involve-
ment of sex, strain and age factors in high fat diet-induced obesity
in C57BL/6J and BALB/cA mice. Exp Anim. 2007;56(4):263-272.
212. Choi JY, McGregor RA, Kwon EY, et al. The metabolic response to a
high-fat diet reveals obesity-prone and -resistant phenotypes in
mice with distinct mRNA-seq transcriptome profiles. Int J Obes
(Lond). 2016;40(9):1452-1460.
213. Svenson KL, Van Smith R, Magnani PA, et al. Multiple trait measure-
ments in 43 inbred mouse strains capture the phenotypic diversity
characteristic of human populations. J Appl Physiol (1985). 2007;
102(6):2369-2378.
214. Kebede MA, Attie AD. Insights into obesity and diabetes at the
intersection of mouse and human genetics. Trends Endocrinol Metab.
2014;25(10):493-501.
215. Tuttle AH, Philip VM, Chesler EJ, Mogil JS. Comparing phenotypic
variation between inbred and outbred mice. Nat Methods. 2018;
15(12):994-996.
216. Ding C, Guo J, Su Z. The status of research into resistance to diet-
induced obesity. Horm Metab Res. 2015;47(6):404-410.
217. Smith RL, Soeters MR, Wust RCI, Houtkooper RH. Metabolic flexi-
bility as an adaptation to energy resources and requirements in
health and disease. Endocr Rev. 2018;39(4):489-517.
218. Bardova K, Horakova O, Janovska P, et al. Early differences in meta-
bolic flexibility between obesity-resistant and obesity-prone mice.
Biochimie. 2016;124:163-170.
219. Poret JM, Souza-Smith F, Marcell SJ, et al. High fat diet consump-
tion differentially affects adipose tissue inflammation and adipocyte
size in obesity-prone and obesity-resistant rats. Int J Obes (Lond).
2018;42(3):535-541.
220. Hong J, Stubbins RE, Smith RR, Harvey AE, Nunez NP. Differential
susceptibility to obesity between male, female and ovariectomized
female mice. Nutr J. 2009;8:11.
221. Giles ED, Jackman MR, MacLean PS. Modeling diet-induced obesity
with obesity-prone rats: implications for studies in females. Front
Nutr. 2016;3:50.
222. Franconi F, Seghieri G, Canu S, Straface E, Campesi I, Malorni W.
Are the available experimental models of type 2 diabetes appropri-
ate for a gender perspective? Pharmacol Res. 2008;57(1):6-18.
223. Parks BW, Sallam T, Mehrabian M, et al. Genetic architecture of
insulin resistance in the mouse. Cell Metab. 2015;21(2):334-347.
224. Wang S, Yehya N, Schadt EE, Wang H, Drake TA, Lusis AJ. Genetic
and genomic analysis of a fat mass trait with complex inheritance
reveals marked sex specificity. PLoS Genet. 2006;2(2):e15.
225. Collaboration NCDRF. Trends in adult body-mass index in 200 coun-
tries from 1975 to 2014: a pooled analysis of 1698 population-
based measurement studies with 19.2 million participants. Lancet.
2016;387(10026):1377-1396.
226. Hoeg LD, Sjoberg KA, Jeppesen J, et al. Lipid-induced insulin resis-
tance affects women less than men and is not accompanied by
inflammation or impaired proximal insulin signaling. Diabetes. 2011;
60(1):64-73.
227. Mascarenhas-Melo F, Marado D, Palavra F, et al. Diabetes abrogates
sex differences and aggravates cardiometabolic risk in postmeno-
pausal women. Cardiovasc Diabetol. 2013;12:61.
PREGUIÇA ET AL.
228. Ter Horst KW, Gilijamse PW, de Weijer BA, et al. Sexual dimorphism
in hepatic, adipose tissue, and peripheral tissue insulin sensitivity in
obese humans. Front Endocrinol (Lausanne). 2015;6:182.
229. Kautzky-Willer A, Harreiter J, Pacini G. Sex and gender differences
in risk, pathophysiology and complications of type 2 diabetes
mellitus. Endocr Rev. 2016;37(3):278-316.
230. Macotela Y, Boucher J, Tran TT, Kahn CR. Sex and depot differences
in adipocyte insulin sensitivity and glucose metabolism. Diabetes.
2009;58(4):803-812.
231. Lee MJ, Wu Y, Fried SK. Adipose tissue heterogeneity: implication
of depot differences in adipose tissue for obesity complications. Mol
Aspects Med. 2013;34(1):1-11.
232. Stubbins RE, Holcomb VB, Hong J, Nunez NP. Estrogen modulates
abdominal adiposity and protects female mice from obesity and
impaired glucose tolerance. Eur J Nutr. 2012;51(7):861-870.
233. Chen X, McClusky R, Chen J, et al. The number of X chromosomes
causes sex differences in adiposity in mice. PLoS Genet. 2012;8(5):
e1002709.
234. Link JC, Hasin-Brumshtein Y, Cantor RM, et al. Diet, gonadal sex,
and sex chromosome complement influence white adipose tissue
miRNA expression. BMC Genomics. 2017;18(1):89.
235. Jackman MR, Kramer RE, MacLean PS, Bessesen DH. Trafficking of
dietary fat in obesity-prone and obesity-resistant rats. Am J Physiol
Endocrinol Metab. 2006;291(5):E1083-E1091.
236. Jackman MR, MacLean PS, Bessesen DH. Energy expenditure in
obesity-prone and obesity-resistant rats before and after the intro-
duction of a high-fat diet. Am J Physiol Regul Integr Comp Physiol.
2010;299(4):R1097-R1105.
237. Nadal-Casellas A, Proenza AM, Llado I, Gianotti M. Sex-dependent
differences in rat hepatic lipid accumulation and insulin sensitivity in
response to diet-induced obesity. Biochem Cell Biol. 2012;90(2):
164-172.
238. Medrikova D, Jilkova ZM, Bardova K, Janovska P, Rossmeisl M,
Kopecky J. Sex differences during the course of diet-induced obe-
sity in mice: adipose tissue expandability and glycemic control. Int J
Obes (Lond). 2012;36(2):262-272.
239. Tokuyama Y, Sturis J, DePaoli AM, et al. Evolution of β-cell dysfunc-
tion in the male Zucker diabetic fatty rat. Diabetes. 1995;44(12):
1447-1457.
240. McCullough LD, de Vries GJ, Miller VM, Becker JB, Sandberg K,
McCarthy MM. NIH initiative to balance sex of animals in preclinical
studies: generative questions to guide policy, implementation, and
metrics. Biol Sex Differ. 2014;5:15.
241. Mauvais-Jarvis F, Arnold AP, Reue K. A guide for the design of pre-
clinical studies on sex differences in metabolism. Cell Metab. 2017;
25(6):1216-1230.
242. Houtkooper RH, Argmann C, Houten SM, et al. The metabolic foot-
print of aging in mice. Sci Rep. 2011;1:134.
243. van der Heijden RA, Bijzet J, Meijers WC, et al. Obesity-induced
chronic inflammation in high fat diet challenged C57BL/6J mice is
associated with acceleration of age-dependent renal amyloidosis. Sci
Rep. 2015;5:16474.
244. Bray GA, Lovejoy JC, Smith SR, et al. The influence of different fats
and fatty acids on obesity, insulin resistance and inflammation.
J Nutr. 2002;132(9):2488-2491.
245. Stanhope KL, Goran MI, Bosy-Westphal A, et al. Pathways and
mechanisms linking dietary components to cardiometabolic disease:
thinking beyond calories. Obes Rev. 2018;19(9):1205-1235.
246. Alvheim AR, Malde MK, Osei-Hyiaman D, et al. Dietary linoleic acid
elevates endogenous 2-AG and anandamide and induces obesity.
Obesity (Silver Spring). 2012;20(10):1984-1994.
247. Ailhaud G, Massiera F, Weill P, Legrand P, Alessandri JM, Guesnet P.
Temporal changes in dietary fats: role of n−6 polyunsaturated fatty
acids in excessive adipose tissue development and relationship to
obesity. Prog Lipid Res. 2006;45(3):203-236.
PREGUIÇA ET AL.
248. Vimaleswaran KS, Berry DJ, Lu C, et al. Causal relationship between
obesity and vitamin D status: bi-directional Mendelian randomiza-
tion analysis of multiple cohorts. PLoS Med. 2013;10(2):e1001383.
249. Showalter MR, Nonnecke EB, Linderholm AL, et al. Obesogenic diets
alter metabolism in mice. PLoS ONE. 2018;13(1):e0190632.
250. Yang SC, Lin SH, Chang JS, Chien YW. High fat diet with a high
monounsaturated fatty acid and polyunsaturated/saturated fatty
acid ratio suppresses body fat accumulation and weight gain in
obese hamsters. Nutrients. 2017;9(10):1148.
251. Takeuchi H, Matsuo T, Tokuyama K, Shimomura Y, Suzuki M. Diet-
induced thermogenesis is lower in rats fed a lard diet than in those
fed a high oleic acid safflower oil diet, a safflower oil diet or a lin-
seed oil diet. J Nutr. 1995;125(4):920-925.
252. Piers LS, Walker KZ, Stoney RM, Soares MJ, O'Dea K. The influence
of the type of dietary fat on postprandial fat oxidation rates: mono-
unsaturated (olive oil) vs saturated fat (cream). Int J Obes Relat
Metab Disord. 2002;26(6):814-821.
253. Jones PJ, Jew S, AbuMweis S. The effect of dietary oleic, linoleic,
and linolenic acids on fat oxidation and energy expenditure in
healthy men. Metabolism. 2008;57(9):1198-1203.
254. Marques C, Mega C, Goncalves A, et al. Sitagliptin prevents inflam-
mation and apoptotic cell death in the kidney of type 2 diabetic ani-
mals. Mediators Inflamm. 2014;2014:538737.
255. Mega C, de Lemos ET, Vala H, et al. Diabetic nephropathy ameliora-
tion by a low-dose sitagliptin in an animal model of type 2 diabetes
(Zucker diabetic fatty rat). Exp Diabetes Res. 2011;2011:162092.
256. Goncalves A, Leal E, Paiva A, et al. Protective effects of the
dipeptidyl peptidase IV inhibitor sitagliptin in the blood-retinal bar-
rier in a type 2 diabetes animal model. Diabetes Obes Metab. 2012;
14(5):454-463.
257. Warden CH, Fisler JS. Comparisons of diets used in animal models
of high-fat feeding. Cell Metab. 2008;7(4):277.
258. Benoit B, Plaisancie P, Awada M, et al. High-fat diet action on adi-
posity, inflammation, and insulin sensitivity depends on the control
low-fat diet. Nutr Res. 2013;33(11):952-960.
259. Chassaing B, Miles-Brown J, Pellizzon M, et al. Lack of soluble fiber
drives diet-induced adiposity in mice. Am J Physiol Gastrointest Liver
Physiol. 2015;309(7):G528-G541.
260. Pellizzon MA, Ricci MR. The common use of improper control diets
in diet-induced metabolic disease research confounds data interpre-
tation: the fiber factor. Nutr Metab (Lond). 2018;15:3.
261. Dalby MJ, Ross AW, Walker AW, Morgan PJ. Dietary uncoupling of
gut microbiota and energy harvesting from obesity and glucose tol-
erance in mice. Cell Rep. 2017;21(6):1521-1533.
262. Ussar S, Griffin NW, Bezy O, et al. Interactions between gut micro-
biota, host genetics and diet modulate the predisposition to obesity
and metabolic syndrome. Cell Metab. 2015;22(3):516-530.
29
263. Ganeshan K, Chawla A. Warming the mouse to model human dis-
eases. Nat Rev Endocrinol. 2017;13(8):458-465.
264. Karp CL. Unstressing intemperate models: how cold stress under-
mines mouse modeling. J Exp Med. 2012;209(6):1069-1074.
265. Cannon B, Nedergaard J. Nonshivering thermogenesis and its ade-
quate measurement in metabolic studies. J Exp Biol. 2011;214(Pt 2):
242-253.
266. Tian XY, Ganeshan K, Hong C, et al. Thermoneutral housing acceler-
ates metabolic inflammation to potentiate atherosclerosis but not
insulin resistance. Cell Metab. 2016;23(1):165-178.
267. Giles DA, Moreno-Fernandez ME, Stankiewicz TE, et al. The-
rmoneutral housing exacerbates nonalcoholic fatty liver disease in
mice and allows for sex-independent disease modeling. Nat Med.
2017;23(7):829-838.
268. Speakman JR, Keijer J. Not so hot: optimal housing temperatures for
mice to mimic the thermal environment of humans. Mol Metab.
2012;2(1):5-9.
269. Kumar Singh A, Cabral C, Kumar R, et al. Beneficial effects of dietary
polyphenols on gut microbiota and strategies to improve delivery
efficiency. Nutrients. 2019;11(9):2216.
270. Fernandes R, Viana SD, Nunes S, Reis F. Diabetic gut microbiota
dysbiosis as an inflammaging and immunosenescence condition that
fosters progression of retinopathy and nephropathy. Biochim
Biophys Acta Mol Basis Dis. 2019;1865(7):1876-1897.
271. Turnbaugh PJ, Ridaura VK, Faith JJ, Rey FE, Knight R, Gordon JI.
The effect of diet on the human gut microbiome: a metagenomic
analysis in humanized gnotobiotic mice. Sci Transl Med. 2009;1(6):
6ra14.
272. David LA, Maurice CF, Carmody RN, et al. Diet rapidly and repro-
ducibly alters the human gut microbiome. Nature. 2014;505(7484):
559-563.
273. Yang Q, Liang Q, Balakrishnan B, Belobrajdic DP, Feng QJ,
Zhang W. Role of dietary nutrients in the modulation of gut micro-
biota: a narrative review. Nutrients. 2020;12(2):381.
274. Wallis KF, Melnyk SB, Miousse IR. Sex-specific effects of dietary
methionine restriction on the intestinal microbiome. Nutrients.
2020;12(3):781.
How to cite this article: Preguiça I, Alves A, Nunes S, et al.
Diet-induced rodent models of obesity-related metabolic
disorders—A guide to a translational perspective. Obesity
Reviews. 2020;1–29. https://doi.org/10.1111/obr.13081