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High-fat, high-sugar diet induces splenomegaly that is ameliorated with exercise and genistein treatment

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Abstract

Objective We tested the effect of exercise training and genistein treatment on splenomegaly in mice fed a high-fat, high-sugar diet (HFSD). Results Male and female C57BL6 mice fed HFSD containing 60% fat along with drinking water containing 42 g/L sugar (55% sucrose/45% fructose) for 12 weeks exhibited significant obesity, hyperglycemia, and elevated plasma IL-6 levels. This was accompanied by splenomegaly characterized by spleen weights 50% larger than mice fed standard chow (P < 0.05) with enlarged rad and white pulps. Mice fed HFSD and treated with a combination of exercise (30 min/day, 5 days/week) and genistein (600 mg genistein/kg diet) had reduced spleen weight (P < 0.05). The decrease in spleen weight was associated with a significant improvement in red-to-white pulp area ratio and plasma glucose and IL-6 (P < 0.05). Our findings indicate that reversal of splenomegaly by regular exercise and genistein treatment may be important in the clinical management of HFSD-induced obesity.
Buchanetal. BMC Res Notes (2018) 11:752
https://doi.org/10.1186/s13104-018-3862-z
RESEARCH NOTE
High-fat, high-sugar diet induces
splenomegaly thatisameliorated withexercise
andgenistein treatment
Levi Buchan1, Chaheyla R. St. Aubin2, Amy L. Fisher2, Austin Hellings1, Monica Castro3, Layla Al‑Nakkash4,
Tom L. Broderick5 and Jeffrey H. Plochocki3,6*
Abstract
Objective: We tested the effect of exercise training and genistein treatment on splenomegaly in mice fed a high‑fat,
high‑sugar diet (HFSD).
Results: Male and female C57BL6 mice fed HFSD containing 60% fat along with drinking water containing 42 g/L
sugar (55% sucrose/45% fructose) for 12 weeks exhibited significant obesity, hyperglycemia, and elevated plasma IL‑6
levels. This was accompanied by splenomegaly characterized by spleen weights 50% larger than mice fed stand‑
ard chow (P < 0.05) with enlarged rad and white pulps. Mice fed HFSD and treated with a combination of exercise
(30 min/day, 5 days/week) and genistein (600 mg genistein/kg diet) had reduced spleen weight (P < 0.05). The
decrease in spleen weight was associated with a significant improvement in red‑to‑white pulp area ratio and plasma
glucose and IL‑6 (P < 0.05). Our findings indicate that reversal of splenomegaly by regular exercise and genistein treat‑
ment may be important in the clinical management of HFSD‑induced obesity.
Keywords: High‑fat diet, High‑sugar diet, Spleen, Exercise, Genistein
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Introduction
Obesity, type 2 diabetes mellitus, and other metabolic
disorders are being reconceptualized as inflammatory
conditions [1, 2]. For example, obesity induced by high-
fat, high-sugar diet (HFSD) is associated with prolonged
elevation of proinflammatory serum markers such as
IL-6 and inflammation in peripheral tissues, as well as
metabolic dysregulation, including insulin and leptin
resistance [3, 4]. Although the effects of diet-induced
metabolic dysregulation and inflammation are widely
documented in many organs, its effects on spleen mor-
phology have yet to be thoroughly characterized.
e spleen is the largest secondary lymphoid organ
in the body and is composed of two functional regions,
white and red pulp. White pulp is lymphoid tissue
containing immune cells that target blood-borne patho-
gens, whereas red pulp is a site of erythrophagocytosis
[5, 6]. Inflammation induced by HFSD has been shown
to modulate splenic function by causing increased phos-
phatidylserine externalization of red blood cells and
thus promoting the interaction with erythrophagocy-
tosis macrophages [6], and by inducing extramedul-
lary hematopoiesis of monocyte-like cells secondary to
inflammation [7]. ese changes in splenic function and
morphology have been implicated in the pathogenesis of
diabetes and obesity-related cardiovascular disease and
kidney disease [6, 8]. erefore, therapeutic modalities
that maintain normal splenic morphology in the obese
condition may prove beneficial to long-term health.
In this study, we examine metabolic and proinflamma-
tory markers, spleen weight, and spleen histomorphome-
try in mice fed a HFSD and treated with either exercise or
the isoflavone genistein, or both. Treatment with exercise
and isoflavones have been shown to ameliorate periph-
eral inflammation through antioxidative actions and by
Open Access
BMC Research Notes
*Correspondence: jeffrey.plochocki@ucf.edu
6 Department of Medical Education, College of Medicine, University
of Central Florida, 6850 Lake Nona Blvd, Orlando, FL 85308, USA
Full list of author information is available at the end of the article
Page 2 of 6
Buchanetal. BMC Res Notes (2018) 11:752
reducing levels of proinflammatory cytokines [3, 9, 10].
In this study, we hypothesize that exercise and genistein
treatment in mice fed a HFSD mitigates diet-induced
changes in spleen weight and morphology.
Main text
Methods
Fifty female and 50 male mice of the strain C57BL6 (Jax
Labs, ME, USA) were used in the study. At the age of
6weeks, the mice were randomly divided into 5 treat-
ment groups of 10 mice per sex. Treatment groups were
assigned as follows: (1) untreated control mice, (2) mice
fed a HFSD, (3) mice fed a HFSD and treated with exer-
cise, (4) mice fed a HFSD and treated with genistein, (5)
and mice fed a HFSD and treated with exercise and gen-
istein. Treatment was administered for 12weeks. Mice in
the HFSD groups were fed pellets with 60% fat, 20% pro-
tein and 20% carbohydrate (Dyets Inc. Bethlehem, PA,
USA) and given 42g/L sugar dissolved in drinking water
(55% fructose/45% sucrose). is diet induces significant
visceral obesity and insulin resistance in the C57BL/6
mouse [11]. Control mice were given standard drinking
water and rodent chow that contained 20.3 g protein,
66g carbohydrate, and 5g fat. All foods and liquids were
administered adlibitum. Exercise treatment consisted of
low-intensity treadmill running for 30min/day, 5days/
week. Exercise of this duration and intensity was chosen
because it has been shown to reduce insulin resistance
in C57BL/6 mice with diet-induced obesity [12]. Gen-
istein treatment was administered at 600mg genistein/
kg HFSD diet (Dyets Inc., PA, USA). We have previously
found this genistein dose incorporated into diet is suf-
ficient to produce significant increases in free genistein
in plasma and to benefit bone and gut health [13, 14].
During the study, mice were housed at a temperature of
22°C with a light/dark period of 12-h. Use of the ani-
mals was approved by the Institutional Animal Care and
Use Committee at Midwestern University. e protocol
of the experiment complied with the National Institutes
of Health’s Guide for the Care and Use of Laboratory
Animals.
Following sacrifice at an age of 4months, spleens were
harvested, weighed, and embedded in paraffin blocks.
Spleens were then sectioned longitudinally in the mid-
line at 5 μm thickness and stained with hematoxylin
and eosin (H&E) for histological evaluation under light
microscopy. ImageJ (v1.6, NIH) was used to measure
the area of the spleen and the ratio of white to red pulp,
calculated as [(red pulp area white pulp area)/white
pulp area]. Enlargement of one or both of these splenic
regions may indicate dysfunction. Plasma was collected
for measurement of glucose (Autokit, Wako Diagnostics,
Richmond, VA, USA), insulin, and IL-6 (Milliplex Assay,
Millipore, Billerica, MA, USA) following the manufac-
turers’ instructions. Two-way ANOVA was used to test
for differences among the treatment groups and between
females and males. Significance was set at P < 0.05. Sta-
tistical analyses were conducted using SPSS Statistics 25
software (IBM, USA).
Results
Food intake measured by weight was similar in mice fed
HFSD and lean mice fed a standard diet (P > 0.05), yet
mice fed HFSD had significantly greater body mass than
lean mice (P < 0.05, Fig. 1). Mice fed HFSD and treated
with genistein had reduced food intake and body mass in
comparison to mice fed HFSD alone (P < 0.05). Analysis
of plasma markers showed mice treated with HFSD had
elevated glucose, insulin, and IL-6 in comparison to lean
mice fed standard diet (P < 0.05, Fig. 1). Treatment with
exercise and genistein in combination reduced plasma
glucose and IL-6 in mice fed HFSD (P < 0.06). When com-
paring males and females within each treatment group,
we found males fed a standard diet had greater body
masses, food intake, and plasma glucose and insulin
levels than females given the same diet (P < 0.05). Male
mice also had higher body mass and insulin levels than
females in every treatment group except HFSD + gen-
istein (P < 0.05).
Comparisons of spleen weights showed mice fed HFSD
had significantly enlarged spleens relative to lean mice
(P < 0.05, Fig.2). However, there was no difference in red-
to-white pulp ratio between these treatment groups, indi-
cating the increase in splenic weight in HFSD mice is due
to expansion of both the red and white pulps (P > 0.05,
Fig.2). Mice fed HFSD and treated with both genistein
and exercise had reduced spleen weight and red-to-
white pulp ratios in comparison to mice fed HFSD alone
(P < 0.05). Male and female mice fed HFSD responded
similarly to treatment with exercise and genistein alone
and in combination (Fig.2). Microscopic examination of
the spleens found increased cellularity in the red pulps
of mice fed a HFSD in comparison to mice fed a stand-
ard diet and HFSD mice treated with exercise and/or
genistein (Fig.3). e red pulps of mice fed HFSD con-
tained numerous macrophages, which were not present
to the same extent in mice of the other treatment groups
(Fig.3).
Discussion
Our results show that HFSD significantly alters splenic
morphology. Mice fed a HFSD exhibited significant
splenic enlargement in comparison to control mice
after 12 weeks of treatment. Given that 6 weeks of
high-fat diet administration in rats does not signifi-
cantly increase spleen weight [15], our findings suggest
Page 3 of 6
Buchanetal. BMC Res Notes (2018) 11:752
doubling the treatment period or the addition of sugar
to the diet may be needed to induce splenomegaly.
We found no significant difference in the ratio of red-
to-white pulp area in mice fed a HFSD. is suggests
splenomegaly is likely attributed to concomitant mor-
phological changes in both the red and white pulp.
Altukkaynak etal. [16] found treatment with high fat
diet causes sinusoids and surrounding tissue to expand
in both the red and white pulps, rather than finding
histological changes specific to one pulp. is is con-
sistent with our microscopic observations of the spleen.
We further observed numerous macrophages adjacent
to the sinusoids of mice fed a HFSD. While HFSD has
yet to be studied in the spleen, administration of a
high fat diet to mice has been shown to enhance eryth-
rophagocytosis by macrophages [6]. Our observation of
increased macrophage presence in the spleen indicates
this also occurs with HFSD.
Fig. 1 Body mass (a), food intake (b), and plasma levels of glucose (c), insulin (d), and IL‑6 (e) by treatment group. *Significant difference between
lean mice fed standard diet and mice fed HFSD (P < 0.05); significant difference with mice fed HFSD (P < 0.05); significant difference between males
and females given the same treatment (P < 0.05). LN, lean mice fed standard diet (n = 10 females, 8 males); HFSD, high‑fat, high‑sugar diet (n = 9
females, 9 males); Ex, exercise (n = 9 females, 10 males); Gen, genistein (n = 8 females, 8 males); GenEx, genistein and exercise (n = 10 females, 8
males). Data are expressed as mean ± 2 SE
Fig. 2 Analysis of spleen weight (a) and ratio of red pulp area to white pulp area of the spleen (b) by sex and treatment group. As the ratio
approaches 0.0, there is greater white pulp area relative to red pulp area. *Significant difference between lean mice fed standard diet and mice
fed HFSD (P < 0.05); significant difference with mice fed HFSD (P < 0.05). LN, lean mice fed standard diet (n = 10 females, 8 males); HFSD, high‑fat,
high‑sugar diet (n = 9 females, 9 males); Ex, exercise (n = 9 females, 10 males); Gen, genistein (n = 8 females, 8 males); GenEx, genistein and exercise
(n = 10 females, 8 males). Data are expressed as mean ± 2 SE
Page 4 of 6
Buchanetal. BMC Res Notes (2018) 11:752
A central goal of our study was to determine the thera-
peutic value of exercise and the isoflavone genistein on
reducing splenomegaly. We show treatment with exer-
cise and genistein in combination inhibits the formation
of splenomegaly in mice fed a HFSD. Genistein inhibits
angiogenesis by inhibiting proliferation of endothelial
cells [17]. It is possible that the anti-angiogenic effects
of genistein prevent sinusoidal dilation in the spleen,
thereby ameliorating splenomegaly associated with
HFSD. is has been hypothesized as an explanation for
how genistein treatment reduces splenomegaly in mice
with malaria-infected red blood cells, and may be acting
in a similar manner here [18]. Genistein also blocks the
ingestion of RBCs by macrophages through its actions as
a tyrosine kinase inhibitor [19]. Erythrophagocytosis is
a major cause of splenomegaly and interruption of this
process in the red pulp by genistein may help explain its
influence on spleen weight in our study. It may also help
explain why the red-to-white pulp ratio is decreased in
mice fed HFSD and treated with genistein and exercise.
However, additional research is required to explicate the
precise mechanism of the contribution of genistein to
splenomegaly prevention.
Splenic volume has been reported to decrease in vol-
ume during exercise, likely from contractile reticular cells
within the splenic stroma [20, 21]. However, this change
appears to be transient, although long-term data are lack-
ing [22]. It is perhaps more likely that the contribution
of exercise to the observed decrease in spleen weight is
associated with reductions in obesity-related inflamma-
tion. Exercise is protective against central obesity and
insulin resistance, and is associated with a reduction in
proinflammatory serum markers [23]. Exercise modu-
lates the function of immune cells that are abundant in
the spleen, namely lymphocytes and macrophages [24,
25]. Even light exercise is sufficient to reduce circulat-
ing proinflammatory cytokines like TNF secreted by
lymphocytes and macrophages [26, 27]. Alterations in
immune cell function, particularly in the obese condition
[28], may contribute to reductions in spleen volume.
Fig. 3 Representative histological sections of the spleen for each treatment group. Note the high cellularity of the control HFSD‑fed mice, which
is not present to the same extent in the HFSD treated with exercise and/or genistein. Mice fed HFSD also have numerous macrophages in the red
pulp (asterisks) in comparison to the other treatment groups. Splenic morphological appearance did not differ by sex. LN, lean mice fed standard
diet (n = 10 females, 8 males); HFSD, high‑fat, high‑sugar diet (n = 9 females, 9 males); Ex, exercise (n = 9 females, 10 males); Gen, genistein (n = 8
females, 8 males); GenEx, genistein and exercise (n = 10 females, 8 males). RP, red pulp; WP, white pulp. Histological analysis was conducted on all
100 mice. H&E stain. Scale bar 50 μm
Page 5 of 6
Buchanetal. BMC Res Notes (2018) 11:752
e efficacy of treatment with a combination of gen-
istein and exercise in the HFSD-fed mice on spleno-
megaly is further corroborated by reductions in body
mass and plasma glucose and IL-6 levels also identified
in this treatment group. ese results suggest genistein
and exercise in combination improve metabolic func-
tion and inhibit inflammation systemically. Treatment
with genistein and exercise in combination may mitigate
splenomegaly by beneficially affecting glucose and IL-6
pathways. Glucose intake induces oxidative stress at the
cellular and molecular levels that causes inflammation
through secretion of IL-6 [29], a proinflammatory marker
associated with the obese condition and complications
such as cardiovascular disease [30, 31]. Cells incubated
with genistein exhibit decreased IL-6 production [32,
33]. IL-6 production is also modulated by glucose cellu-
lar uptake during exercise [34, 35]. e combined effects
of genistein and exercise on glucose uptake and IL-6
expression may be responsible for the reduction in spleen
weight noted in the current study, and may likely have
benefits beyond the spleen. However, additional data are
needed to elucidate the precise effects of genistein and
exercise treatment in the HFSD-induced obese condition.
In summary, this study presents novel findings that
augment the current understanding of how splenic mor-
phology is influenced by diet. We show that, (1) HFSD
administered over a 12-week period is sufficient to cause
splenomegaly in mice, (2) combined exercise and gen-
istein treatment may reverse splenic enlargement associ-
ated with HFSD, and (3) the reduction in splenomegaly
with combined exercise and genistein treatment directly
correlates with reductions in plasma glucose and IL-6
levels. ese findings may have implications for the treat-
ment of inflammation and splenomegaly associated with
HFSD diet.
Limitations
Our study is limited in several ways. To better define
changes to splenic pulp cellular composition, immuno-
histochemistry should be conducted to identify prolifera-
tion in immune cell populations. Red blood cell labeling
in conjunction with a splenic phagocytosis assay would
also better inform our understanding of alterations in
erythrophagocytosis for each treatment. Interpretations
of our findings should be qualified by these limitations.
Abbreviations
HFSD: high‑fat, high sugar diet; Ex: exercise; Gn: genistein; RP: red pulp; WP:
white pulp.
Authors’ contributions
LA, TLB, and JHP contributed to planning the experiments. AF, CS exercise
trained the mice for the 12 week study. AF, CS, LB, AH, MC, LA, and JHP
conducted the experimental analyses. LB and JHP drafted the manuscript.
All other authors edited and revised the manuscript. LA, TLB, and JHP were
responsible for securing the funding. All authors read and approved the final
manuscript.
Author details
1 Arizona College of Osteopathic Medicine, Midwestern University, Glendale,
AZ, USA. 2 College of Graduate Studies, Midwestern University, Glendale, AZ,
USA. 3 Department of Anatomy, College of Graduate Studies and Arizona
College of Osteopathic Medicine, Midwestern University, Glendale, AZ, USA.
4 Department of Physiology, College of Graduate Studies and Arizona College
of Osteopathic Medicine, Midwestern University, Glendale, AZ, USA. 5 Depart‑
ment of Physiology, Laboratory of Diabetes and Exercise Metabolism, College
of Graduate Studies and Arizona College of Osteopathic Medicine, Midwest‑
ern University, Glendale, AZ, USA. 6 Department of Medical Education, College
of Medicine, University of Central Florida, 6850 Lake Nona Blvd, Orlando, FL
85308, USA.
Acknowledgements
We wish to acknowledge the Arizona Alzheimer’s Consortium (LA, TLB, JHP)
and Midwestern University Intramural Funds (LA, TLB, and JHP) for funding our
research.
Competing interests
The authors declare that they have no competing interests.
Availability of data and materials
The datasets used in the current study are available from the corresponding
author by request.
Consent for publication
Not applicable.
Ethics approval
The protocol for this study was approved by the Midwestern University Institu‑
tion Animal Care and Use Committee (IACUC Protocol #2880).
Funding
Midwestern University and Arizona Alzheimer’s Consortium funded the study.
Publisher’s Note
Springer Nature remains neutral with regard to jurisdictional claims in pub‑
lished maps and institutional affiliations.
Received: 6 August 2018 Accepted: 17 October 2018
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... In another study, quantitative analysis of histological tissue staining and fluorescence was investigated using this software [14]. Using this software, changes in spleen tissue resulting from a high-fat and highsugar diet and its treatment with exercise were studied [15]. In another study, image analysis was used for detecting diabetes by examining tomographic images of light-emitting bodies [16]. ...
... Another study focused on quantitative analysis of histological and fluorescence staining using this software [14]. Additionally, changes in the tissue structure of the spleen due to a high-fat and highcarbohydrate diet and its treatment with exercise were examined with ImageJ software [15]. The results of the current study confirmed the high accuracy and capabilities of this software in image analysis of tissue sections from meat products, which aligns with previous studies. ...
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In recent years, there has been a growing interest in methods for assessing the percentage of meat (skeletal muscle) in meat products. Given the high margin of error in methods such as chemical analysis, the most reliable and accurate approach for assessing the percentage of skeletal muscle in meat products is histology and subsequent use of image analysis. Due to limited research in this field and the not-so-easy access to some image analysis software, the present study, for the first time, examines the percentage of skeletal muscle in meat products and the time spent on analyzing each sample using two freely accessible graphic software programs (Adobe Photoshop and ImageJ) and two non-free graphic software programs (Clemex and Image Pro-Plus). For this purpose, 100 samples of meat products (30 Kielbasa, 30 sausages, 20 hamburgers, 10 kebab bite, and 10 chicken nuggets) with a known skeletal muscle content were used. After transferring the samples to the laboratory and preparing tissue sections using the Hematoxylin-Eosin staining method, the images of tissue sections were analyzed using the mentioned software programs. The results showed almost equal accuracy of all four software programs assessing skeletal muscles. However, the time required to analyze each ImageJ sample was significantly lower than the other software programs (p< 0.05). Based on the results of this study, it appears that ImageJ software offers greater competence for image analysis of tissue sections and determining the percentage of skeletal muscle in meat products.
... При ЦД підвищується рівень запальних факторів, спричинених гіперглікемією, а також рівень оксидативного стресу, що призводить до апоптозу клітин селезінки та імунної дисфункції, які, у свою чергу, зумовлюють пошкодження селезінки [24,31]. Крім того, активна робота поліолового шляху призводить до надмірного використання НАДФН, а виснаження НАДФН, в свою чергу, призводить до обмеження здатності глутатіонредуктази відновлювати рівень головного внутрішньоклітинного антиоксиданту -глутатіону, що спричиняє його зниження, підвищення АФК, зміну профілю цитокінів і, зрештою, порушення роботи імунної системи [4]. ...
... Хронічні ускладнення при ЦД 2 типу пов'язані зі збільшенням кількості лейкоцитів, навіть якщо вони не перевищують норму. У ряді досліджень виявлено різноманітні гістопатологічні змі ни селезінки, включаючи атрофію білої пульпи та розширення червоної пульпи при моделюванні ЦД у піддослідних тва-рин [20,31,32]. Згідно з даними різних досліджень, в макрофагах спостерігалося значне відкладення гемосидерину порівняно з контрольними групами [32,33]. ...
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Diabetes mellitus is an important medical problem that leads to a large number of complications. As is widely known, the immune system is highly susceptible in cases of diabetes, resulting in diverse infectious com- plications. The study of changes in the immune system in diabetes mellitus is important for understanding their pathogenesis and developing effective methods of treatment and prevention of this disease. The purpose of the research is to establish the degree of study of the patterns of morpho-functional chan- ges in the spleen and the immune system as a whole in diabetes according to the literature. Material and methods. Study and generalization of local and foreign literature, results of meta-analyses and randomized trials. The literature search for the review was carried out using PubMed, SeinceDirect, Europa PMC, BMC, MedLine, etc. databases. Results. A study of the literature shows that the main mechanisms of diabetic damage to the immune system are the result of oxidative stress and the development of chronic inflammation in tissues. Additionally, as pro-inflammatory cytokines, adhesion molecules, and chemokines, result in endoplasmic reticulum impair- ment and mitochondrial stress, ultimately initiating cellular programmed cell death and contributing to tissue damage and immune system disruption. With the progression of the disease, characteristic histopathological changes were observed in the spleen, namely: atrophy of the white pulp, fibrous changes in the capsule and tra- becular expansion, and increased apoptosis of the splenocytes themselves. At the cellular level, one of the key roles in maintaining chronic inflammation is played by an imbalance of Treg and Th17 cells, decreased natural killer (NK) cell activity, and changes in the M1/M2 ratio, which shifts towards pro-inflammatory macrophages. Conclusions. Significant progress has been made in the study of changes in the immune system in dia- betes over the past decade It has been proven that during diabetes there is an effect of oxidative stress on the immune system at all levels, from cellular to organ. This effect is realized, first of all, through the formation of an imbalance in the regulatory mechanisms of immunity and the initiation of chronic inflammation and cell apoptosis, which are leading factors in the development of specific and non-specific diabetic complications. K ey wor d s : diabetes mellitus, oxidative stress, immune system, immune system damage, review.
... 22 Furthermore, the simultaneous presence of HFD and LPS administration appears to have a synergistic impact on dyslipidemia and endotoxemia, as both factors can independently lead to gut barrier disruption 23,24 and liver dysfunction. 25,26 These findings suggest that HFD consumption can cause metabolic disruptions and inflammation, which can be further exacerbated by LPS administration. In addition, exogenous LPS administration contributes to splenomegaly by stimulating the immune system, resulting in the production of white blood cells and an increase in spleen size, 27 whereas chronic HFD consumption increases serum LPS levels and free fatty acids, 26 which subsequently trigger peripheral immune cells and fat cell responses, resulting in the production of inflammatory cytokines such as IL-1β. ...
... 25,26 These findings suggest that HFD consumption can cause metabolic disruptions and inflammation, which can be further exacerbated by LPS administration. In addition, exogenous LPS administration contributes to splenomegaly by stimulating the immune system, resulting in the production of white blood cells and an increase in spleen size, 27 whereas chronic HFD consumption increases serum LPS levels and free fatty acids, 26 which subsequently trigger peripheral immune cells and fat cell responses, resulting in the production of inflammatory cytokines such as IL-1β. 28 Overall, these findings suggest that HFD and LPS may jointly amplify inflammatory responses, and LPS can exacerbate inflammation in the context of HFD-induced obesity. ...
Article
Aim: Prolonged high-fat diet (HFD) consumption has been shown to impair cognition and depression. The combined effects of HFD and lipopolysaccharide (LPS) administration on those outcomes have never been thoroughly investigated. This study investigated the effects of LPS, HFD consumption, and a combination of both conditions on microglial dysfunction, microglial morphological alterations, synaptic loss, cognitive dysfunction, and depressive-like behaviors. Methods: Sixty-four male Wistar rats were fed either a normal diet (ND) or HFD for 12 weeks, followed by single dose-subcutaneous injection of either vehicle or LPS. Then, cognitive function and depressive-like behaviors were assessed. Then, rats were euthanized, and the whole brain, hippocampus, and spleen were collected for further investigation, including western blot analysis, qRT-PCR, immunofluorescence staining, and brain metabolome determination. Results: HFD-fed rats developed obese characteristics. Both HFD-fed rats with vehicle and ND-fed rats with LPS increased cholesterol and serum LPS levels, which were exacerbated in HFD-fed rats with LPS. HFD consumption, but not LPS injection, caused oxidative stress, blood-brain barrier disruption, and decreased neurogenesis. Both HFD and LPS administration triggered an increase in inflammatory genes on microglia and astrocytes, increased c1q colocalization with microglia, and increased dendritic spine loss, which were exacerbated in the combined conditions. Both HFD and LPS altered neurotransmitters and disrupted brain metabolism. Interestingly, HFD consumption, but not LPS, induced cognitive decline, whereas both conditions individually induced depressive-like behaviors, which were exacerbated in the combined conditions. Conclusions: Our findings suggest that LPS aggravates metabolic disturbances, neuroinflammation, microglial synaptic engulfment, and depressive-like behaviors in obese rats.
... The metabolic and phenotypic data from mice in all five groups were reported [24,25]. Mice fed an HFSD displayed obesity, hyperinsulinemia, hyperglycemia, and T2DM [24,25]. ...
... The metabolic and phenotypic data from mice in all five groups were reported [24,25]. Mice fed an HFSD displayed obesity, hyperinsulinemia, hyperglycemia, and T2DM [24,25]. Figure 1 illustrates the significant differences in plasma IL-6, chondrocyte MMP-13, and col10a expression in lean mice and mice fed an HFSD. Figure 2 displays immunofluorescence staining of MMP-13 and col10a in articular cartilage of the proximal tibia. ...
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Type 2 diabetes mellitus and osteoarthritis (OA) often present as comorbidities. We examined the role of plasma IL-6, chondrocyte MMP-13, and col10a expression in the development of OA in obese diabetic mice. We further investigated dietary genistein and exercise training as potential mitigators of OA. One hundred adult mice (50 females, 50 males) aged 6 weeks were randomized into 5 groups, including lean controls, obese diabetic controls, and obese diabetic mice treated with genistein, exercise training, and genistein plus exercise training. The obese diabetic state was induced by feeding the mice a high-fat, high-sugar diet. Genistein was incorporated into the diet at a concentration of 600 mg genistein/kg. Exercise training was performed on a treadmill and consisted of daily 30 min sessions at 12 m/min, 5 days/week for a 12-week period. After treatment, plasma was collected, and proximal tibias were removed for analysis. Plasma IL-6 and MMP-13 were elevated while col10a was reduced in obese diabetic mice in comparison to lean controls. Dietary genistein treatment reduced IL-6 and MMP-13 expression and increased col10a expression. Histological examination of articular cartilage showed reduced thickness of the uncalcified zones and proteoglycan content in the cartilage of diabetic mice in comparison to mice fed genistein. Exercise training had no significant effect. In conclusion, genistein (and not exercise training) attenuates OA by reducing IL-6 and MMP-13 expression in diabetic mice.
... Immunotoxicity studies report that immune system suppressant substances increase spleen size, i.e., splenomegaly, as during urethane intervention (Robles 2014). Buchan et al. (2018) demonstrated that animals with splenomegaly induced by a high-fat diet showed significant spleen size reduction with physical exercise and genistein (isoflavones) interventions. Thus, it seems that C. sessiliflora may have a potential regulatory effect on splenic function and morphology due to its biochemical composition (Castro et al. 2020;Castro et al. 2022). ...
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Campomanesia sessiliflora (O.Berg) Mattos is a Brazilian native plant species used in a popular medicinal tea for treating gastrointestinal, urinary, and dermatological pathologies. This study evaluated the toxicity of Campomanesia sessiliflora (O.Berg) Mattos via acute and subacute toxicity tests. It also analyzed mutagenic and genotoxic potentials by the micronucleus test, which detects genetic material damage indicating mutagenicity, and the comet assay, which assesses DNA damage levels as a genotoxicity indicator. The plant extract initially originated from the ultrasonic maceration of Campomanesia sessiliflora (O.Berg) Mattos leaves in a hydroethanolic solution. The involved animals were adult Wistar rats. Ten females were available to evaluate acute toxicity and estimate the LD50, receiving a dose of 2000 mg/kg. The subacute toxicity evaluation used 35 females and 35 males divided into seven groups: negative control (saline control – SC), positive control (cyclophosphamide control – CC), 125 mg/kg (125), 250 mg/kg (250), 500 mg/kg (500), 1000 mg/kg (1000), and the satellite group (ST). Genotoxicity and mutagenicity experiments applied bone marrow micronucleus and comet assays. Acute and subacute toxicity tests did not present behavioral, physical, and physiological changes (p≥0.05). Administering the Campomanesia sessiliflora (O.Berg) Mattos extract reduced spleen size in male and female animals, without histopathological changes. However, doses above 500 mg/kg showed significant genotoxic and mutagenic effects in the comet and micronucleus assays compared to the control group. The extract did not exhibit acute or subacute toxicity, but doses higher than 500 mg/kg indicated some level of genotoxicity and mutagenicity.
... The impacts of folate deficiency and the HFF diet on systemic immune responses were evaluated by the functions of splenocytes. Our results showed that C57BL/6 mice fed the HFF diet had significantly larger spleen compared to those fed a normal-fat diet, which was similar to another study [41]. The spleen is the largest peripheral lymphoid organ in the body, containing a large number of immune cells such as T and B cells that secret cytokines and modulate the systemic immune response. ...
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The prevalence of obesity and chronic kidney disease (CKD) is increasing simultaneously and rapidly worldwide. Our previous study showed that folate deficiency increased lipid accumulation and leptin production of adipocytes. Whether folate plays a role in CKD, particularly obesity-related nephropathy remains unclear. To investigate the effects of folate deficiency on CKD in diet-induced obese mice, four groups of male C57BL/6 mice were fed either a normal-fat diet (NF) with folate (NF+f); NF without folate (NF−f); high-fat high-fructose diet (HFF) with folate (HFF+f); or HFF without folate (HFF−f) for 12 months during the study. The results showed that HFF increased not only body weight, fasting blood glucose, total cholesterol (TC), low-density lipoprotein (LDL)-cholesterol, and blood pressure, but also cytokines levels, such as interleukin (IL)-2, interferon (IFN)-γ, IL-17A/F, IL-6, monocyte chemoattractant protein (MCP)-1, and transforming growth factor (TGF)-β1. The indicators of kidney failure including urinary protein, neutrophil gelatinase-associated lipocalin (NGAL), renal type I and IV collagen deposits and leptin content, and serum creatinine were also increased by HFF. Folate-deficient diets further elevated serum TC, LDL-cholesterol, IL-6, tumor necrosis factor (TNF)-α, MCP-1, TGF-β1, and leptin, but decreased IL-10 level, and thus exacerbated renal fibrosis. To investigate the possible mechanisms of folate deficiency on renal injury, phosphorylation of pro-fibrosis signaling molecules, including signal transducer and activator of transcription (STAT)3 and small mothers against decapentaplegic (Smad)2/3, were assayed. Both HFF and folate deficiency significantly increased the phosphorylation of STAT3 and Smad2/3, suggesting synergistic effects of HFF−f on chronic renal inflammation and fibrosis. In conclusion, the results demonstrated that folate deficiency might aggravate inflammatory status and enhance renal fibrosis.
... Genistein (4,5,7-trihydroxyisoflavone) is one of the main isoflavones distributed in the germ of soybean, fava bean, and alfalfa, whose physiological actions have been widely confirmed in humans and animals. In addition to improving immunity in mammals fed with high-carbohydrate and high-fat diets, genistein has been shown to exert effects on oxidative stress and lipid and glucose metabolism [26][27][28][29][30]. Additionally, studies have indicated that the control of obesity by diets rich in soy isoflavones is mainly achieved by reducing blood triglyceride (TG) and total cholesterol (T-CHO) in broiler breeder hens and rabbits [31,32]. ...
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A 56-day feeding trial was conducted to investigate the effects of genistein on growth, lipid metabolism, antioxidant capacity, and immunity of common carp fed with high-carbohydrate or high-fat diets. Five diets were used to feed fish: control diet (5% fat; CO), high-fat diet (11% fat; HF), high-carbohydrate diet (45% carbohydrate; HC), and HF or HC diet with 500 mg/kg genistein (FG or CG). Results showed that final body weight (FW) and specific growth rate (SGR) were significantly reduced, but the supplementation with genistein resulted in higher values of FW and SGR than the HF or HC group. Both high carbohydrate and high fat belong to high-energy diets, which may promote lipid deposition. Genistein obviously decreased liver triglyceride (TG) content and alleviated hepatic fat vacuolation in the HF and HC groups. The expression of lipid metabolism genes (cpt-1 and atgl) was markedly higher in the FG group than in the HF group. The lipid synthesis-related genes (fas, acc, and pparγ) were elevated in high-energy diets but recovered to the control level or reduced after genistein treatments. With respect to fatty acid transporter genes, fatp increased in the FG group, and cd36 increased in the CG group. Furthermore, the antioxidant and immune indexes, such as total antioxidant capacity (T-AOC), glutathione peroxidase (GSH-PX), superoxide dismutase (SOD), acid phosphatase (ACP), and lysozyme (LZM) activities, were decreased, while malonate aldehyde (MDA) content, activities of alanine aminotransferase (ALT), and aspartate aminotransferase (AST) were enhanced in the HF and HC groups. The antioxidant and immunity values could be ameliorated by treatment with genistein. Moreover, the transcript levels of antioxidant-related genes (cat, gr, and nrf2) in the liver and anti-inflammatory factors (tgf-β and il-10) and lyz in the head kidney tissue were promoted, although the expression levels of proinflammatory factors (tnf-α and il-6) declined in the genistein supplementation group, which confirmed the antioxidant and immune-enhancing effects of genistein. Therefore, 500 mg/kg genistein could ameliorate the negative effects of high-energy diets on immunity.
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Understanding sex differences in immunological responses in the context of obesity is important to improve health outcomes. This systematic review aimed to investigate sex differences in systemic inflammation, immune cell phenotype, and function in diet‐induced obesity (DIO) animal models. A systematic search in Medline, Embase, and CINAHL from inception to April 2023 was conducted, using a combination of the following concepts: sex, obesity, cytokines, and immune cell phenotypes/function. Forty‐one publications reporting on systemic inflammation (61%), cell phenotype (44%), and/or function (7%) were included. Females had lower systemic inflammation compared with males in response to DIO intervention and a higher proportion of macrophage (M)2‐like cells compared with males that had a higher proportion of M1‐like in adipose tissue. Although there were no clear sex differences in immune function, high‐fat DIO intervention remains an important factor in the development of immune dysfunction in both males and females, including disturbances in cytokine production, proliferation, and migration of immune cells. Yet, the mechanistic links between diet and obesity on such immune dysfunction remain unclear. Future studies should investigate the role of diet and obesity in the functionality of immune cells and employ adequate methods for a high‐quality investigation of sex differences in this context.
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A high-fat diet induces obesity in mice, leading to insulin resistance, decreased mitochondrial function, and increased apoptosis in the hippocampus, which eventually result in memory loss. The present study investigated the effect of physical exercise on memory, hippocampal mitochondrial function, and apoptosis in mice with in insulin resistance caused by obesity due to high-fat diet. Mice were randomly divided into four groups: control (CON), control and exercise (CON + EX), high fat diet (HFD), and high fat diet and exercise (HFD + EX). After receiving a high-fat (60%) diet for 20 weeks to induce obesity, the animals were subjected to an exercise routine 6 times per week, for 12 weeks. The exercise duration and intensity gradually increased over 4-week intervals. Hippocampal memory was examined using the step-down avoidance task. Mitochondrial function and apoptosis were also examined in the hippocampus and dentate gyrus. We found that obesity owing to a high-fat diet induced insulin resistance and caused a decrease in memory function. Insulin resistance also caused a decrease in mitochondrial function in the hippocampus by reducing Ca2+ retention and O2, respiration, increasing the levels of H2O2, and Cyp-D, and mPTP opening. In addition, apoptosis in the hippocampus increased owing to decreased expression of Bcl-2 and increased expression of Bax, cytochrome c, and caspase-3 and TUNEL-positive cells. In contrast, physical exercise led to reduced insulin resistance, improved mitochondrial function, and reduced apoptosis in the hippocampus. The results suggest that physiological stimulations such as exercise improve hippocampal function and suppress apoptosis, potentially preventing the memory loss associated with obesity-induced insulin resistance.
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Background Obese, type two diabetics are at an increased risk for fracturing their limb bones in comparison to the general population. Phytoestrogens like as the soy isoflavone genistein have been shown to protect against bone loss. In this study, we tested the effects of genistein treatment on femurs of ob/ob mice, a model for obesity and type two diabetes mellitus. Methods Twenty six-week-old female mice were divided into obese (ob/ob) control, obese genistein-treated, lean (ob/+) control, and lean genistein-treated groups (n = 5 each). Treatment with genistein consisted of 600 mg genistein/kg diet. Control mice were given standard rodent chow. At the end of a four-week treatment period, bone histomorphometric and three-point bending properties were compared among groups. ResultsObese mice had larger bone areas (B.Ar.; P < 0.05) and total areas (Tt.Ar.; P < 0.05), but similar bone volume (B.Ar./Tt.Ar.; P > 0.05) of the proximal femoral epiphysis in comparison to lean mice. Treatment with genistein decreased Tt.Ar. and femur length, and increased ultimate force required to fracture the femur and the maximum deformation to failure (P < 0.05). Conclusions Genistein improves resistance to fracture from bending loads.
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Inflammation, a biological response of body tissues to harmful stimuli, is also known to be involved in a host of diseases, such as obesity, atherosclerosis, rheumatoid arthritis, and even cancer. Isoflavones are a class of flavonoids that exhibit antioxidant, anticancer, antimicrobial, and anti-inflammatory properties. Increasing evidence has highlighted the potential for isoflavones to prevent the chronic diseases in which inflammation plays a key role, though the underlying mechanisms remain unclear. Recently, some studies have raised concerns about isoflavones induced negative effects like carcinogenesis, thymic involution, and immunosuppression. Therefore, this review aims to summarize the anti-inflammatory effects of isoflavones, unravel the underlying mechanisms, and present the potential health risks.
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Background Obesity and type 2 diabetes mellitus are associated with elevated risk of limb bone fracture. Incidences of these conditions are on the rise worldwide. Genistein, a phytoestrogen, has been shown by several studies to demonstrate bone-protective properties and may improve bone health in obese type 2 diabetics. Methods In this study, we test the effects of genistein treatment on limb bone and growth plate cartilage histomorphometry in obese, hyperglycemic ob/ob mice. Six-week-old ob/ob mice were divided into control and genistein-treated groups. Genistein-treated mice were fed a diet containing 600 mg genistein/kg for a period of 4 weeks. Cross-sectional geometric and histomorphometric analyses were conducted on tibias. Results Genistein-treated mice remained obese and hyperglycemic. However, histomorphometric comparisons show that genistein-treated mice have greater tibial midshaft diameters and ratios of cortical bone to total tissue area than the controls. Genistein-treated mice also exhibit decreased growth plate thickness of the proximal tibia. Conclusion Our results indicate that genistein treatment affects bone of the tibial midshaft in the ob/ob mouse, independent of improvements in the hyperglycemic state and body weight.
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Background: -High fat diet (HFD) promotes endothelial dysfunction and pro-inflammatory monocyte activation, which contribute to atherosclerosis in obesity. We investigated whether HFD also induces dysfunction of red blood cells (RBC), which serve as a reservoir for chemokines via binding to Duffy antigen receptor for chemokines (DARC). Methods and results: -60% HFD for 12 weeks, which produced only minor changes in lipid profile in C57/BL6 mice, markedly augmented the levels of MCP-1 bound to RBC, which in turn stimulated macrophage migration through an endothelial monolayer. Levels of RBC bound KC were also increased by HFD. These effects of HFD were abolished in DARC -/- mice. In RBC from HFD-fed wild-type and DARC -/- mice, levels of membrane cholesterol and phosphatidylserine externalization were increased, fostering RBC-macrophage inflammatory interactions and promoting macrophage phagocytosis in vitro. When labeled ex vivo and injected into control mice, RBC from HFD-fed mice exhibited ~3 fold increase in splenic uptake. Finally, RBC from HFD-fed mice induced increased macrophage adhesion to the endothelium when they were incubated with isolated aortic segments, indicating endothelial activation. Conclusions: -RBC dysfunction, analogous to endothelial dysfunction, occurs early during diet-induced obesity and may serve as a mediator of atherosclerosis. These findings may have implications for the pathogenesis of atherosclerosis in obesity, a worldwide epidemic.
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Genistein, a phytoestrogen that demonstrates bone‐protective properties, is currently being investigated as a treatment for a number of conditions that affect skeletal health, including type 2 diabetes. Type 2 diabetics have an elevated risk of developing fractures, particularly in bones of the lower limb. Type 2 diabetics also tend to be obese ‐ as many as 85% according to the World Health Organization. This study investigates the effects of genistein treatment on the properties of lower limb bones in obese, diabetic mice. Twenty mice of the ob/ob strain aged 6 weeks were randomly divided into two groups. One group received 600 mg/kg of genistein in their chow for 4 weeks. Comparisons of tibial midshafts show mice treated with genistein have significantly greater diameters and ratios of cortical bone to total tissue area (B.ar/Tt.Ar) than controls (P < 0.05). Evaluation of the proximal tibial growth plate reveals significantly decreased growth plate thickness in genistein‐treated mice, suggesting genistein affects endochondral ossification. Although these initial findings are promising, further study is needed to assess the effects of genistein on fracture risk in type 2 diabetics.
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High fat diet-induced obesity is associated with insulin resistance (IR) and other chronic, diet related illnesses, including dementia. Alzheimer disease is the most common form of dementia, and is characterized by the presence of amyloid plaques and neurofibrillary tangles in brain. This study was designed to determine whether diet-induced changes in peripheral insulin sensitivity could contribute to alterations in brain insulin signaling and cognitive functions. Four week old, male C57BL/6NHsd mice were randomly assigned a high fat diet (40% energy from fat) with 42 g/L liquid sugar (HFS) added to the drinking water or a normal chow diet (12% energy from fat) for 14 weeks. Metabolic phenotypes were characterized for energy expenditure, physical activity, and food intake, and glucose and insulin tolerance tests. In addition, we examined the changes in protein expression related to brain insulin signaling and cognitive function. Mice fed HFS exhibited a statistically significant increase in obesity, and lower glucose and insulin tolerance as compared to animals fed the normal chow diet. In brain, HFS elicited IR as evidenced by a significant decrease in tyrosine phosphorylation of insulin receptor and an increase serine phosphorylation of IRS-1. These changes were accompanied by inflammatory (NFκB, JNK) and stress responses (p38 MAPK and CHOP) in whole brain lysate. In addition, HFS mouse brain exhibited biochemical changes related to increased amyloid beta deposition and neurofibrillary tangle formation, and decreased synaptic plasticity. These results suggested changes in insulin sensitivity might contribute to cognitive impairment associated with the HFS diet in mice.
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Recent data have revealed that the plasma concentration of inflammatory mediators, such as tumour necrosis factor-α (TNF-α) and interleukin-6 (IL-6), is increased in the insulin resistant states of obesity and type 2 diabetes, raising questions about the mechanisms underlying inflammation in these two conditions. It is also intriguing that an increase in inflammatory mediators or indices predicts the future development of obesity and diabetes. Two mechanisms might be involved in the pathogenesis of inflammation. Firstly, glucose and macronutrient intake causes oxidative stress and inflammatory changes. Chronic overnutrition (obesity) might thus be a proinflammatory state with oxidative stress. Secondly, the increased concentrations of TNF-α and IL-6, associated with obesity and type 2 diabetes, might interfere with insulin action by suppressing insulin signal transduction. This might interfere with the anti-inflammatory effect of insulin, which in turn might promote inflammation.