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

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.

Full text

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
© The Author(s) 2018. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License
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and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creat iveco mmons .org/
publi cdoma in/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
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

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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

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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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