Dietary supplementation with Apis mellifera wholemeal flour

reduces hepatic steatosis in obese mice

Running Head: Bee wholemeal flour improves steatosis

Aline L. Nascimento1, Joyce H. S Pereira2, Bruna V. Caldas1, Victor H. D.

Guimarães2, Renato S. Monteiro-Junior2, Alfredo M. B. Paula2, André L. S.
Guimarães2, Ulisses A. Pereira1, Sérgio H. S. Santos2
1
Laboratório Instituto de Ciências Agrárias (ICA), Postgraduate Program in Food and
Health, Universidade Federal de Minas Gerais (UFMG), Montes Claros, Minas Gerais,
Brazil; 2Laboratory of Health Sciences, Postgraduate Program in Health Sciences,
Universidade Estadual de Montes Claros (UNIMONTES), Montes Claros, Minas Gerais,
Brazil.

ABSTRACT: Chronic diseases associated with metabolism are becoming one of the most prevalent health problems in the world.
Therefore, searching for foods with functional properties is a viable strategy to prevent the onset of chronic diseases. Amongst the
most currently studied functional foods, the edible insects stand out, and Apis mellifera (bee) has received prominent attention due
to its high nutritional value. In this context, the present study aimed to evaluate the effects of a food supplementation with Apis
mellifera wholemeal flour on the mice's metabolic profile with diet-induced hepatic steatosis. Six-week-old male Swiss mice were
divided into 6 groups and treated for 4 weeks. The groups were identified as follows: standard diet (AIN93), standard diet + 15%
Apis mellifera (AIN93+15%), standard diet + 30% Apis mellifera (AIN93+30%), hyperlipidic diet (HFD), and, hyperlipidic diet +
15% Apis mellifera (HFD+15%), hyperlipidic diet + 30% wholemeal bee Apis mellifera flour (HFD+30 %). Several liver
parameters such as liver histology, tissue weight, and liver mRNA expression were evaluated. The main results showed that the
inclusion of Apis mellifera wholemeal flour reduced hepatic steatosis by improving biochemical and histological parameters.
Moreover, significant changes were also observed in the expression of lipogenic genes PPARγ, SRBP1c, and GPX4. The main
findings showed the potential use of Apis mellifera wholemeal flour in improving hepatic steatosis and its beneficial effects on
metabolic diseases.

KEYWORDS: • Metabolic syndrome . hepatic steatosis . edible insects . bee

INTRODUCTION

As defined by the World Health Organization
(WHO), metabolic syndrome (MS) is a sum of
inflammatory diseases characterized by insulin
resistance and dyslipidemia, with risk factors
interrelated with obesity 1. Free fatty acids (FFA) are
energy molecules that play an important role in
metabolism, however, excessive circulating free
fatty acids are stored as triglycerides in adipocytes
or ectopically in different organs 2. Chronic
metabolic diseases are becoming a pandemic,
increasing the global public health risk by increasing
type 2 diabetes mellitus (DM2), obesity, and non-
alcoholic fatty liver disease (NAFLD) 3.
Non-alcoholic fatty liver disease (NAFLD) is
considered the most common liver disorder in the
world and its chronic state can progress to liver
fibrosis or cirrhosis inducing up to liver
transplantation, and/or death 4. Oxidative stress is
considered one of the main mechanisms of liver
damage and progression of NAFLD, associated with
poor diet, a sedentary lifestyle, and other external
factors 5. The pursuit for quality of life, and
adherence to healthy habits, including weight loss
and structured exercise, remains the master key to
treating and preventing NAFLD 2.
Several studies have used experimental animal
models to examine natural product substances such
as bioactive compounds, proteins, carbohydrates,
dietary fibers, lipids, and vitamins for their health
benefits improving NAFLD 6. Some studies support
the understanding of a synergistic effect, between
lifestyle interventions and the so-called functional
foods 7. The price and scarcity of animal-source
foods associated with the increased demand for
proteins and high nutritional and functional food
value have lately encouraged the practice of insect
consumption (entomophagy).
In Africa, several insects are an important source
of protein and provide food security. Among the
most consumed insect orders in the world are the
Coleoptera (beetles), Isoptera (termites), Diptera
(flies), and the increasing consumption of
Hymenoptera (ants, bees, and wasps) 8,9,10. Among
the edible insects, the bee wholemeal (Apis
mellifera) has a high nutritional content and
promotes health benefits. Recent studies using mice
models to evaluate the effects of bee powder and
extract consumption showed the potential of bees
inducing immunomodulatory and anti-inflammatory
activities 11,12,13. In this context, the present study
aimed to evaluate the effect of adding bee
wholemeal flour in a hyperlipidic diet-induced
NAFLD mice model.
MATERIALS AND METHODS
Animals
The experiment was performed with 36 6-week-
old male Swiss mice from the Central Animal
Facility of the Federal University of Minas Gerais.
Six mice were placed in each cage and kept under
controlled conditions of luminosity with a 12-hour
light/dark cycle and temperature of 22°C±2°C,
besides free access to water and diet during
treatment. All procedures were performed following
the Ethics Committee on Animal Experimentation
and Welfare of the State University of Montes
Claros (UNIMONTES), protocol number 224/2021
14.
Experimental Diets
The study consisted of 6 experimental groups,
comprising the following diets: Standard diet
(AIN93); Standard diet + 15% bee wholemeal flour
(AIN93+15); Standard diet + 30% bee wholemeal
flour (AIN93+30); Hyperlipidic diet (HFD);
Hyperlipidic diet + 15% bee wholemeal flour
(HFD+F15) and Hyperlipidic diet + 30% Apis
mellifera bee wholemeal flour (HFD+F30). The
AIN93-M diet was designed according to
experiments conducted by the previous study12,
while the hyperlipidic diet was developed according
to Guimaraes et. al. 15. After the 2 months, the
groups with induced obesity were added with bee
wholemeal flour for the next 1 month of treatment.
All groups with the Apis mellifera wholemeal
inclusion were isocaloric to their respective controls
14,15
.
Bromatological analysis
The bees were purchased from the company
Apiário Terra Nova, Montes Claros-MG at the adult
stage. Afterward, they were dehydrated in an SSD
CR oven at a temperature of 60 °C for 24 hours 15.
After drying, the bees were taken to the electric
grinder to obtain the flour (Bottini 1/3cv, Brazil).
The flour was homogenized and after the moisture
test, a sample was taken at the Faro Veterinary
Laboratory for bromatological analysis.
Biochemical Analysis
After the euthanasia of the animals, the blood
was collected and taken to a centrifuge at 3200 rpm
for 10 minutes at 4 °C to obtain serum. The enzymes
alanine transaminases (AST/GOT) and aspartate
transferase (ALT/GPT) were dosed using enzyme
kits (Wiener Laboratorios, Rosario, Argentina)
16,17,18
. (Measurements were performed in Wiener
BT-3000 plus Chemical Analyzer equipment
(Wiener®, Argentina).
Histology
Liver samples were fixed, embedded, sectioned,
and stained with hematoxylin and eosin (H&E) for
morphological analysis by routine histological
methods. Images of the white adipose tissue slides
(x10 ocular lens and x40 objective lens) were
obtained with an FSX100 inverted microscope
(Olympus®, Japan). The area of adipocytes was
calculated in five representative fields of each slide
18-19
.
Real-time quantitative PCR
Total RNA was extracted from liver samples
using Trizol reagent (Invitrogen Corp.®, USA).
Extracted RNA was treated with DNAse
(Promega®, USA) and cDNA was obtained by
reverse transcription using M-MLV (Invitrogen
Corp.®, USA), and random hexamer primers. The
qRT-PCR analysis was performed with SYBR
Green PCR Master Mix (Applied Biosystems®,
USA) in a Quant Studio 6 flex, 384-well equipment
(Applied Biosystems®, USA). Relative gene
expression levels were normalized against the
control glyceraldehyde 3-phosphate dehydrogenase
(GAPDH) gene and analyzed using the 2-ΔΔCt.
method. The sequence of the primer used in the
study 17 (Table 1).
Data Analysis
 Normality was verified using the Shapiro-Wilk
test. Data were analyzed using GraphPad Prism
software, (version 5.0®, San Diego, California,
USA) and expressed as means±standard error of the
mean (SEM), with 95% (p< 0.05) confidence.
Comparison between two groups was performed
using Student's t-test and multiple comparisons were
performed using one-way ANOVA or two-way
ANOVA tests, followed by Bonferroni post-test.

RESULTS

Liver analysis

To evaluate the effects of Apis mellifera

wholemeal on serum transaminases, GPT, GOT, and
GPT/GOT ratio were tested as shown in (Fig 2C). Fig. 1. (A) Liver weight; *p<0.05; ANOVA; BW: AIN93:
standard diet; AIN93 + F15: standard diet with 15%
For the evaluation of the effects on systemic organs, wholemeal Apis mellifera; AIN93 + F30: standard diet with
the liver was chosen because it is a vascularized 30% wholemeal Apis mellifera; HFD: hypercaloric diet; HFD
organ that favors the observation of various + F15: hypercaloric diet with 15% wholemeal Apis mellifera;
metabolic changes. GOT levels tended to decrease in HFD + F30: hypercaloric diet with 30% whole Apis
the groups treated with AIN93 + F15 growth mellifera. Biochemical profile of mice fed standard and
hyperlipidic diets. (B) GOT/ALT levels. (C) GPT/AST
(14.29±1.86 U/L) and AIN93 + F30 growth
levels. (D) GOT/GPT ratio (U/L).*p<0.05; **p<0.01,
(13.71±1.75 U/L) when compared with the groups ANOVA ## p<0. 01 T-test; GOT/AST: aspartate
treated with AIN93 Growth control (19.17±2.72 aminotransferase; GPT/ALT: alanine aminotransferase;
U/L). Whereas HFD + F15 group (12.86±2.13 U/L) AIN93: standard diet; AIN93 + F15: standard diet with 15%
showed a significant reduction when compared to wholemeal Apis mellifera; AIN93 Growth + F30: standard
the HFD group (23.50±1.47 U/L) (p<0.01) and HFD with 30% wholemeal Apis mellifera; DAP + F15:
hypercaloric diet with 15% wholemeal Apis mellifera; DAP +
+ F30 (17.14±1.35 U/L) (p<0.01) as shown in (Fig.
F30: hypercaloric diet with 30% wholemeal Apis mellifera.
2A).
Histological analysis
The liver data for the evaluation of enzyme
transaminases showed a trend of reduction in AIN93
Seeking to better understand the effects of Apis
Growth (91.50±13.01 U/L) and AIN93 Growth +
mellifera, histological analyses were performed as
F30 (95.67±7.18 U/L) groups when compared with
presented in (Fig 3). Among the standard diet
AIN93G + F15 group (99.50±13.98 U/L). However,
groups, there was no significant difference in fat
HFD + F15 group (65.50±10.47 U/L) showed a
deposition in hepatocytes AIN93 (0.00±0.00 μm2);
statistically different reduction from the HDF group
AIN93 + F15 (3327±1024 μm2); AIN93 + F30
(65.50±10.47 U/L) (p<0.01) and HFD + F30 group
(13950±3360 μm2) (Fig. 3A). In turn, the group’s
(97.33±11.09 U/L), for TGP levels, presented in
hypercaloric diet had a significant increase in fat
(Fig. 2B).
deposition among hepatocytes (Fig. 3B). However,
the introduction of bee meal significantly decreased
The GOT/GPT ratio was reduced in AIN93
fat deposition in HFD + F15 (15680±7401 μm2)
Growth + F15 (0.10±0.01 U/L) and AIN93 Growth
(p<0.01); HFD + F30 (9238±7479 μm2) (p<0.01)
+ F30 (0.12±0.01 U/L) when compared with its
groups compared with their control HFD
control group AIN93 Growth (0.14±0.02 U/L) and
(103900±34260 μm2) groups (Fig. 3B).
showed a significant reduction in HFD + F15 (0.
16±0.03 U/L) (p< 0.05) compared to its control
group HFD (0.27±0.02 U/L) and trend of reduction
compared to HFD + F30 group (0.15±0.01 U/L)
(p<0.05) depicted in (Fig. 2 C).

Fig. 2. Histological evaluation of the liver in H&E
staining of mice fed standard and hyperlipidic diets. (A)
Macroscopic and histological images of liver tissue; (B) Area
of fat droplets in liver tissue (μm2).**p<0. 01, ANOVA;
AIN93 standard diet; AIN93 Growth + F15: standard diet
with 15% wholemeal Apis mellifera; AIN93+ F30 standard
diet with 30% wholemeal Apis mellifera; HFD diet:
hypercaloric diet; HFD + F15: hypercaloric diet with 15%
wholemeal Apis mellifera; HFD + F30: hypercaloric diet with
30% wholemeal Apis mellifera.
PPARγ, SREBP1c ,GPX4 mRNA expression
Consumption of Apis mellifera wholemeal flour
modulated gene expression, as shown in (Fig 4).
PPARγ gene expression was significantly decreased
in the AIN93 group (0.67±0.06 A.U.) (p<0.05) and
AIN 93 + 30 group (2.43±0.31 A.U.) (p<0.001)
compared to AIN93 +15 group (4.33±2.25 A.U.). In
the groups fed a hyperlipidic diet there was a
significant reduction in PPARg expression in HFD +
15 (1.09’0.25 A.U.) (p<0.05) and HFD + 30
(0.81±0.0 A.U.) (p<0.01) compared to HFD group
(2.16±0.40 A.U.) (Fig. 4A). SREBP1c gene
expression showed lower significance in the AIN93
control (2.72±0.74 A.U.) (p<0.01 ) and the AIN93 +
30 group (0.41±0.12 A.U.) (p<0.01) compared with
the AIN93 + F15 group (3.60±1.59 A.U.) In the
groups of mice fed a hyperlipidic diet, significantly
higher SREBP1c expression was observed in the
HFD group (3.63±1.01 A.U.) compared with HFD +
F15 (3.63±1.01 A.U.) (p<0.05) and the HFD+30
group (0.77±0.15 A.U.) (p<0.01) (Fig. 4B). The
GPX4 gene showed a trend of lower expression in
the AIN93 + 15 (0.58±0.13 A.U.) and AIN93 + 30
(0.74±0.15 A.U.) treated groups compared to the
AIN93 group. However, the DAF + 15 (0.74±0.15
A.U.) and DAF (0.74±0.15 A.U.) groups showed a
lower expression trend compared to the DAF + F30
group (0.38±0.07 A.U.) as shown in (Fig. 4C).
Fig. 3. Liver gene mRNA expression in mice fed standard
and hyperlipidic diets. (A) mRNA expression of PPARγ. (B)
mRNA expression of SRBP1c. (C) mRNA expression of
GPX4. values are expressed by significance related between
groups. P<0.05, p<0.01, ANOVA ### p<0.0001 T-test. ST
AIN93: standard diet AIN93+F15: a standard diet with 15%
wholemeal Apis mellifera; AIN93+F30: standard with 30%
wholemeal Apis mellifera; HFD: hypercaloric diet;
HFD+F15: hypercaloric diet with 15% wholemeal Apis
mellifera; HFD+F30: hypercaloric diet with 30% wholemeal
Apis mellifera.
DISCUSSION
Dietary supplementation with Apis mellifera (bee
flour) has produced important health benefits in the
liver of obese animals 18,19. Recent studies showed
the ability of insect flour to reduce dyslipidemia by
improving metabolic parameters20. The main
findings of the present research showed the Apis
mellifera wholemeal flour's effects on improving
hepatic steatosis (NAFLD) and modulating the gene
expression related to lipogenesis. The study data
showed an expressive reduction in hepatic fat
deposition in animals receiving bee flour, modifying
the hepatic genes PPARγ, SRBP1c, and GPX4
mRNA.
The used diet was isocaloric when compared
with the respective control after the addition of Apis
mellifera flour (AIN93, AIN93+15, AIN93+30, and
HFD, HFD+15, HFD+30) since all diets have the
same macronutrients content (carbohydrates,
proteins, and lipids). Casein was replaced by insect
flour as the main protein source. Therefore, the
quality of the insect protein measured by the types of
amino acids present in the mice diets can partially
justify the better metabolic results, characterizing as
a factor offering better health benefits 21,22.
The main data showed decreased liver size after
treatment with Apis mellifera and a reduction in liver
weight, consequently decreasing hepatic steatosis.
Hepatic steatosis triggers a variety of changes that
produce an inflammatory condition causing liver
damage, also characterized by the dosage of liver
enzymes GOT and GPT 16,22,23. The development of
hepatic steatosis was evidenced in the groups of
mice receiving the hyperlipidic diet as also
previously demonstrated 17,23-24. However fat
deposition in the adipocytes of the HFD groups
indicated that prolonged feeding can also cause
hypercholesterolemia, 24.
In addition, we evaluated the expression of
lipogenic genes in liver tissue. PPARγ is an
important regulator of lipid and glucose metabolism
in various cell types, thus becoming nuclear
receptors used as targets for drug development to
treat metabolic diseases, including hepatic steatosis
25
. The groups AIN93+30, HFD+15, and HFD+30
showed lower expression making it evident that
these groups presented improved lipid profiles. This
lower expression can be justified by the
consumption of Apis mellifera in the adult stage with
elevated antioxidant activity, given the high
nutritional value, i.e. high protein mineral content
and less fatty acids 26.
The SREBP1c is an important marker related to
fat synthesis being a transcription factor activated in
lipogenic tissues. We observed increased mRNA
levels of SREBP1c in AIN93+15 and HFD groups.
Studies have shown that increased hepatic steatosis
may be related to increased expression of the sterol
regulatory element binding protein 27, 28. GPX4 plays
a key role in protecting cells from free radical
damage and is an antioxidant in cellular protection
against oxidative stress damage. GPX4 levels were
increased in the AIN93 and HFD control groups,
promoting greater cell protection and lower
expression in the groups containing Apis mellifera
flour, indicating that the inclusion of insect flour
promotes an improvement in inflammation caused
by hepatic steatosis 29,30.
In conclusion, the main data of the present study
showed that Apis mellifera wholemeal flour used as
a food source may be a helpful ingredient to a
healthy functional diet for improving metabolic and
hepatic disorders.
ACKNOWLEDGMENTS
This work was partially financed by the
Coordenação de Aperfeiçoamento do Pessoal de
Nível Superior (CAPES), Conselho Nacional de
Desenvolvimento Científico e Tecnológico (CNPq)
and Fundação de Amparo à Pesquisa do Estado de
Minas Gerais (FAPEMIG).
AUTHOR DISCLOSURE STATEMENT
No competing financial interests exist
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