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Citation: Phuong-Nguyen, K.; O’Hely,
M.; Kowalski, G.M.; McGee, S.L.;
Aston-Mourney, K.; Connor, T.;
Mahmood, M.Q.; Rivera, L.R. The
Impact of Yoyo Dieting and Resistant
Starch on Weight Loss and Gut
Microbiome in C57Bl/6 Mice.
Nutrients 2024,16, 3138. https://
doi.org/10.3390/nu16183138
Academic Editors: Henry J.
Thompson and Juscelino Tovar
Received: 31 July 2024
Revised: 5 September 2024
Accepted: 13 September 2024
Published: 17 September 2024
Copyright: © 2024 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
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4.0/).
nutrients
Article
The Impact of Yoyo Dieting and Resistant Starch on Weight
Loss and Gut Microbiome in C57Bl/6 Mice
Kate Phuong-Nguyen 1, 2, * , Martin O’Hely 1,3 , Greg M. Kowalski 2,4, Sean L. McGee 1,2,
Kathryn Aston-Mourney 1,2, Timothy Connor 1,2, Malik Q. Mahmood 5and Leni R. Rivera 1,2 ,*
1School of Medicine, Institute for Mental and Physical Health and Clinical Translation, Deakin University,
Geelong, VIC 3220, Australia; martin.ohely@deakin.edu.au (M.O.); sean.mcgee@deakin.edu.au (S.L.M.);
k.astonmourney@deakin.edu.au (K.A.-M.); timothy.connor@deakin.edu.au (T.C.)
2Metabolic Research Unit, School of Medicine, Deakin University, Waurn Ponds, VIC 3216, Australia;
greg.kowalski@deakin.edu.au
3Murdoch Children’s Research Institute, Royal Children’s Hospital, The University of Melbourne,
Parkville, VIC 3052, Australia
4School of Exercise and Nutrition Sciences, Institute for Physical Activity and Nutrition, Deakin University,
Waurn Ponds, VIC 3216, Australia
5School of Medicine, Deakin University, Waurn Ponds, VIC 3216, Australia; malik.mahmood@deakin.edu.au
*Correspondence: nguyenkate@deakin.edu.au (K.P.-N.); leni.rivera@deakin.edu.au (L.R.R.)
Abstract: Cyclic weight loss and subsequent regain after dieting and non-dieting periods, a phe-
nomenon termed yoyo dieting, places individuals at greater risk of metabolic complications and alters
gut microbiome composition. Resistant starch (RS) improves gut health and systemic metabolism.
This study aimed to investigate the effect of yoyo dieting and RS on the metabolism and gut micro-
biome. C57BL/6 mice were assigned to 6 diets for 20 weeks, including control, high fat (HF), yoyo
(alternating HF and control diets every 5 weeks), control with RS, HF with RS, and yoyo with RS.
Metabolic outcomes and microbiota profiling using 16S rRNA sequencing were examined. Yoyo
dieting resulted in short–term weight loss, which led to improved liver health and insulin tolerance
but also a greater rate of weight gain compared to continuous HF feeding, as well as a different
microbiota profile that was in an intermediate configuration between the control and HF states. Mice
fed HF and yoyo diets supplemented with RS gained less weight than those fed without RS. RS
supplementation in yoyo mice appeared to shift the gut microbiota composition closer to the control
state. In conclusion, yoyo dieting leads to obesity relapse, and increased RS intake reduces weight
gain and might help prevent rapid weight regain via gut microbiome restoration.
Keywords: weight cycling; yoyo dieting; obesity; resistant starch; gut microbiome; metabolism;
short-chain fatty acid; fatty liver
1. Introduction
Overweight and obesity have tripled worldwide since 1975, affecting more than
1.9 billion adults across all ages and socioeconomic groups. This places many people
at a high risk of developing an array of comorbidities, including psychological health
burdens [
1
] and numerous related metabolic disorders [
2
–
6
], such as cardiovascular dis-
eases [
7
,
8
], type 2 diabetes [
9
], and fatty liver diseases [
10
,
11
]. Moreover, obesity has been
associated with lowered life expectancy [
12
] and quality of life [
13
] and has become a major
health and economic crisis in both developed and developing countries [14–17].
For many individuals, weight loss is difficult and often unsustainable. Current ev-
idence suggests that 80–95% of individuals who had previously lost weight typically
regained most of their lost weight within 5 years [
18
–
35
]. Repeated phases of dieting
and non-dieting periods resulting in cyclic weight loss and regain are commonly called
yoyo dieting [
36
]. Currently, weight loss remains the gold standard strategy for many
Nutrients 2024,16, 3138. https://doi.org/10.3390/nu16183138 https://www.mdpi.com/journal/nutrients
Nutrients 2024,16, 3138 2 of 30
metabolic complications, such as diabetes and fatty liver disease [
37
,
38
]. Yoyo dieting
results in relapsing metabolic complications that can surpass the pre-weight loss metabolic
derangement [39,40].
There has been substantial progress in studying the pathogenesis of obesity, especially
with increasing evidence that gut microbiome alterations play an important role in its devel-
opment. However, the role of the gut microbiome in yoyo dieting remains unknown [
41
,
42
].
Gut dysbiosis refers to an imbalance in gut microbiota homeostasis that leads to the loss of
beneficial microbial organisms and changes in gut microbiota composition and function [
43
].
This is known to influence metabolic homeostasis and is associated with an increased risk
of metabolic disturbances [
44
,
45
], including obesity, as shown in both animal [
46
–
48
] and
human studies [
49
–
53
]. Moreover, it is well established that diet is one of the main drivers
of the composition and function of the gut microbiome. Changes in diet profoundly impact
the gut microbiome within days [
54
–
56
]. There is some evidence suggesting that yoyo
dieting results in long–term modification of the gut microbiome profile [
47
]. This alteration
in gut microbiota composition is believed to contribute to susceptibility to weight gain
relapse, hence the challenge of maintaining weight loss [47].
Prior studies suggest that increasing the intake of resistant starch (RS), a type of dietary
fibre, improves gut health [
57
–
64
] and weight management [
65
–
69
]. RS is a type of dietary
fibre that resists digestion in the small intestine and is passed to the large intestine for
fermentation by the colonic microbiome, producing short-chain fatty acids (SCFAs) [
70
].
These SCFAs are known to provide an energy supply for the colonic mucosa [
71
–
73
] and act
as signalling molecules that regulate metabolic pathways [74–85]. The most abundant SC-
FAs are butyrate, acetate, and propionate, which are associated with reduced adiposity [
86
]
and a decreased risk of developing cardiometabolic disease and bowel disorders [
87
–
90
].
RS can be classified into five categories based on composition and source [
60
,
91
–
95
]. This
study investigated the role of RS4, which represents chemically modified starch that is
commercially available as a food ingredient [
95
]. Our study aimed to determine the effects
of yoyo dieting on gut and metabolic health and investigate whether supplementation with
RS might be a promising and effective weight management strategy.
2. Materials and Methods
2.1. Animals and Diets
Five-week-old male and female C57BL/6 mice were purchased from the Animal
Resource Centre (Perth, WA, Australia). Following arrival at our facility, the mice were
acclimatised for 1 week, during which they were fed a normal chow diet. Following this
acclimation period, mice were exposed to six diet treatments (n= 7–8/diet group/sex) for
20 weeks. The sample size was calculated using power calculations based on previous
experiments that examined changes in gastrointestinal function in obesity using a 2-tailed
hypothesis with an alpha of 0.05 and a power of 0.8. Dietary groups included: (1) control
(SF17-091; 12.3% kcal from fat); (2) high fat (HF, SF16-048; 60% kcal from fat); (3) yoyo,
in which mice alternated between an HF and control every 5 weeks (yoyo); (4) control
supplemented with RS (control RS, SF21-019, 13.9% kcal from fat); (5) HF supplemented
with RS (HF RS) (SF21-018, 60% kcal from fat); and (6) yoyo supplemented with RS in the
second weight gain cycle, in which mice were fed an HF (SF16-048) diet for 5 weeks, control
(SF17-091) diet for 5 weeks, HF RS (SF21-018) diet for 5 weeks, and a control (SF17-091) diet
for 5 weeks (yoyo RS) (Figure 1).
Six weeks of age was chosen as the starting age given the increased appearance of
obesity in humans at a corresponding age (mice aged 6–26 weeks correspond to humans
aged approximately 18–32 years old [
96
,
97
]). Mice were housed in groups of 4 per cage and
2 cages per diet treatment, and they were maintained on a strict 12-hour light/dark cycle,
controlled temperature at 21
◦
C, humidity between 40–70% and given ad libitum access to
irradicated pallet food and autoclaved water.
Nutrients 2024,16, 3138 3 of 30
Nutrients 2024, 16, x FOR PEER REVIEW 3 of 31
Figure 1. Diet treatments. Male and female mice were exposed to 6 diets for 20 weeks, including
combinations of control and high-fat (HF) diets with and without resistant starch (RS).
Six weeks of age was chosen as the starting age given the increased appearance of
obesity in humans at a corresponding age (mice aged 6–26 weeks correspond to humans
aged approximately 18–32 years old [96,97]). Mice were housed in groups of 4 per cage
and 2 cages per diet treatment, and they were maintained on a strict 12–hour light/dark
cycle, controlled temperature at 21 °C, humidity between 40–70% and given ad libitum
access to irradicated pallet food and autoclaved water.
All diets were customised by Specialty Feeds Australia (Glen Forrest, Western Aus-
tralia). The RS was supplemented with GemStar® RS, Hamburg, IA, USA (RS type 4, 117–
140 g/kg).
There was no significant difference in the body weight of all animals at the beginning
of the diet intervention. At baseline, mice weighed 20.5 ± 0.5 g (males) or 17 ± 0.5 g (fe-
males). There was no difference in food intake (in grams) between all dietary groups when
measured during weeks 15–17.
All procedures were conducted according to the guidelines of the National Health
and Medical Research Council and approved by the Deakin University Animal Ethics
Commiee. Animals were humanely culled from the study if they showed clinical illness
or experienced 20% body weight loss over 7 days.
Body Weight and Sample Collection: Body weight measurements and faecal collec-
tion were performed weekly throughout the 20-week experimental period. Mice were hu-
manely killed by cervical dislocation after 20 weeks. Terminal cardiac blood was collected
for SCFA analysis, and liver and adipose tissues (gonadal and perirenal) were collected
and weighed.
2.2. Oral Glucose Tolerance Test
To assess glucose homeostasis under physiologically relevant conditions, an oral glu-
cose tolerance test (OGTT) was conducted in week 19 of the study after a 5-hour fast. Glu-
cose (2 g/kg body weight) was administrated by oral gavage in saline (200–300 µL), and
Figure 1. Diet treatments. Male and female mice were exposed to 6 diets for 20 weeks, including
combinations of control and high-fat (HF) diets with and without resistant starch (RS).
All diets were customised by Specialty Feeds Australia (Glen Forrest, Western Australia).
The RS was supplemented with GemStar
®
RS, Hamburg, IA, USA (RS type 4, 117–140 g/kg).
There was no significant difference in the body weight of all animals at the beginning
of the diet intervention. At baseline, mice weighed 20.5
±
0.5 g (males) or 17
±
0.5 g
(females). There was no difference in food intake (in grams) between all dietary groups
when measured during weeks 15–17.
All procedures were conducted according to the guidelines of the National Health
and Medical Research Council and approved by the Deakin University Animal Ethics
Committee. Animals were humanely culled from the study if they showed clinical illness
or experienced 20% body weight loss over 7 days.
Body Weight and Sample Collection: Body weight measurements and faecal collection
were performed weekly throughout the 20-week experimental period. Mice were humanely
killed by cervical dislocation after 20 weeks. Terminal cardiac blood was collected for SCFA
analysis, and liver and adipose tissues (gonadal and perirenal) were collected and weighed.
2.2. Oral Glucose Tolerance Test
To assess glucose homeostasis under physiologically relevant conditions, an oral
glucose tolerance test (OGTT) was conducted in week 19 of the study after a 5-hour fast.
Glucose (2 g/kg body weight) was administrated by oral gavage in saline (200–300
µ
L),
and tail blood was collected at 0, 15, 30, 45, 60, and 90 min post gavage for glucose level
examination (AccuChek, Roche Diagnostics, Indianapolis, IN, USA) and insulin response.
Insulin was determined using a mouse ultrasensitive insulin ELISA kit (ALPCO Diagnostics,
Salem, NH, USA) measurement according to the manufacturer’s protocol.
2.3. Insulin Tolerance Test
An insulin tolerance test (ITT) was conducted in week 20 of the study. Animals
were fasted for 5 h and then administrated 1.0 U/kg body weight Humulin Insulin 30/70
(Eli Lilly) via intraperitoneal injection. Blood glucose measurements were performed 0,
Nutrients 2024,16, 3138 4 of 30
30, 60, 90, and 120 min following the Humulin injection (AccuChek, Roche Diagnostics,
Indianapolis, IN, USA).
2.4. Liver Histology
Liver was fixed in 10% neutral buffered formalin, processed, paraffin-embedded, and
sectioned (5
µ
m) before haematoxylin and eosin staining to assess steatohepatitis. Slides
were examined and photographed using an EVOSTM M7000 Imaging System (AMF7000,
Thermal Fisher Scientific, Waltham, MA USA). Hepatocyte ballooning was defined as
regions of liver cells that had enlarged 1.5–2 times the normal hepatocyte diameter [
98
,
99
].
Hepatocyte ballooning was quantified using 20 randomised light microscopic fields per
animal at ×20 magnification in a blinded fashion.
2.5. Liver Triglyceride Determination
Triglyceride was extracted from 20 mg of frozen mouse liver using Jouihan’s liver
triglyceride assay protocol [
100
]. Prior to the triglyceride assay, the sample was diluted
in sterile Milli-Q water with a ratio of 1/4 sample/water. The free glycerol concentration
was determined in the assay using Triglycerides GPO-PAP (Cobas C pack green, Roche
Diagnostics, Indianapolis, IN, USA). The hepatic triglyceride content was determined using
the following equation:
Triglyceride content (mg/g of liver) = [glycerol] (mg/dL) ×0.0083 (dL)/liver mass (g)
2.6. Short-Chain Fatty Acids (SCFAs)
Plasma propionate and butyrate measurements were made using negative chemical
ionization (NCI) gas chromatography–mass spectrometry (GC–MS, Agilent Technologies,
Santa Clara, CA, USA), based on the method of Tomcik et al. [
101
], with some modifications
described below.
Terminal blood plasma was used for the determination of relative propionate and
butyrate levels. Briefly, 40
µ
L of either plasma or extraction blanks (Milli-Q Water) was
added to a 1 mL glass VEREXTM vial (Phenomenex, Torrance, CA, USA), followed by
the addition of 60
µ
L of Dullbecco modified PBS (no calcium, no magnesium, pH 7.0–7.3
cell culture grade; Thermo Fisher Scientific) containing internal standards (4
µ
M 13C3
propionate and 4
µ
M 13C4 butyrate; Cambridge Isotope Laboratories, Inc., Tewksbury,
MA, USA). The samples were then chemically derivatised via the addition of 200
µ
L of
GC grade acetone containing 100 mM 2,3,4,5,6-pentafluorobenzyl bromide (PFBBr; Sigma,
St. Louis, MO, USA), and after capping the samples were vortexed for 1 min and incubated
at 60
◦
C for 1 h. Subsequently, vials were left to cool down to room temperature, and the
PFB propionate and butyrate esters were extracted via the addition of 125
µ
L of analytical-
grade isooctane, followed by 1 min of vortexing and centrifugation at 5000
×
gfor 5 min.
Finally, 50
µ
L of the top isooctane (organic) phase was removed and transferred to glass
inserts held in 2 mL GC vials, ready for GC–MS analysis. The samples were analysed using
an Agilent 7890B GC system and an Agilent 5977B MSD (Agilent Technologies, Santa Clara,
CA, USA) in NCI mode, with helium as the carrier and methane as the reagent gas. Each
sample was injected (1
µ
L) with a 10:1 split ratio. A VF-5 capillary column with a 10 m inert
EZ-guard (J&W Scientific, Folsom, CA, USA, 30 m, 0.25 mm, 0.25
µ
M) was used, and the
front inlet and transfer line temperatures were set to 250
◦
C and 270
◦
C, respectively, while
the quadrupole and source temperatures were both set to 150
◦
C. The oven temperature
gradient was set to 70
◦
C (1 min), 70
◦
C–160
◦
C at 5
◦
C/min, and 160–320
◦
C at 50
◦
C/min,
followed by a 1-minute hold time at 320
◦
C. The propionate was analysed by selected
monitoring of the 73 m/z (M0; unlabelled) and 76 m/z (M+3; internal standard) ions,
while butyrate was analysed by selected monitoring of the 87 m/z (M0; unlabelled) and
91 m/z (M+4; internal standard) ions. All chromatographic peaks were integrated using the
Quantitative Mass Hunter Workstation (Agilent Technologies, Santa Clara, CA, USA), and
individual sample unlabelled-to-labelled peak area ratios were normalised to the control
group, with data presented as a fold change compared to the control.
Nutrients 2024,16, 3138 5 of 30
2.7. Metabolic Statistical Analysis
Metabolic statistical analysis has been conducted using GraphPad Prism (version
Prism 10.0.2), R (version 2023-03-15), and RStudio (version 2023.06.1+524). Descriptive
and analytical statistics were performed using GraphPad Prism for multiple endpoints,
such as body weight, OGTT, ITT, insulin, and SCFAs, which were analysed using two-way
ANOVA (diet and RS status) with Tukey’s honestly significant difference (HSD) test for
post hoc comparisons unless otherwise stated. If a two-way ANOVA for diet by RS status
indicated a significant interaction between the basic diet type and RS supplementation,
subsequent comparisons of individual diets were evaluated using Tukey’s HSD test. The
rate of weight gain data was analysed in R and RStudio using a linear mixed effect model
with the function lmer from the lme4 package (version 1.1.-35.3) and the lmerTest package
(version 3.1-3) to provide the p-value. Models were fitted using the REML criterium, and
p-values came from t-tests using Satterthwaite’s method. This analysis was conducted to
compare the average rate of body weight regain. The analysis included random and fixed
effects; the random effect was the per-mouse intercept, while the fixed effects were the
slope in different phases (phases 1 and 2), time points within the phase (5 weeks per phase),
and diet/sex groups (4 diets, 2 sexes). Data are presented as mean
±
SEM unless otherwise
stated, with a p-value threshold of 0.05 for statistical significance.
2.8. Taxonomic Microbiota Analysis
Faecal samples collected in weeks 15 and 20 were processed for microbiota analysis.
DNA was isolated using QIAamp Fast DNA Stool Mini kits (Qiagen Pty Ltd., Dandenong,
VIC, Australia), according to the manufacturer’s instructions. Faecal DNA was used for
PCR amplification sequencing (16S rRNA producing amplicon sequence variants (ASVs),
variable region V3–V4), conducted by the Australian Genome Research Facility (Melbourne,
VIC, Australia). Raw data of the metagenome sequencing have been submitted to NCBI
Sequence Read Archive database under submission ID SUB14694204.
Calculations of alpha diversity (Shannon and Fisher) were conducted to assess the micro-
biome diversity within each sample using the estimate_richness function in the phyloseq package
and visualised using the ggplot2 package. Beta diversity was measured using Bray–Curtis
dissimilarities and visualised using principal coordinate analysis (PCoA) plots with phyloseq.
To identify differences in the bacterial microbiota between groups, permutational multivariate
analysis of variance (PERMANOVA) was conducted on the Bray–Curtis dissimilarity matrix
with the adonis2 function within the vegan package. Additionally, DESeq2 was used to deter-
mine genera that were differentially expressed (with a false discovery rate [FDR] of 0.01 to
determine significance) between samples in the phyloseq package.
3. Results
3.1. Body Weight Change, Rate of Weight Regain, and Tissue Mass
3.1.1. Body Weight Change
At the end of the diet intervention, regardless of sex, yoyo mice (green) had a similar
body weight change to control mice (blue) (two-way ANOVA diet
×
RS status in both
sexes p< 0.0001; Tukey’s HSD: male p= 0.8110 (Figure 2A); female p= 0.9995 (Figure 2B))
and were significantly lighter than HF mice (red) (Tukey’s HSD: male p< 0.0001, female
p= 0.0026 (Figure 2)).
Supplementation with RS in a control diet (control RS, purple) did not alter weight
gain in male or female mice in comparison with a control diet only (Tukey’s HSD: male
p> 0.9999 (Figure 2A), female p= 0.9994 (Figure 2B)). In contrast, RS supplementation
in an HF diet (HF RS) (yellow) resulted in significantly less weight gain than an HF diet
alone in male (Tukey’s HSD: p= 0.0002 (Figure 2A)) but not in female mice (Tukey’s HSD:
p= 0.9995 (Figure 2B)).
With RS supplementation in the second phase of the yoyo diet (yoyo RS) (black), there
was no significant difference in the final body weight change compared to the yoyo group
(Tukey’s HSD: both sexes p> 0.9999 (Figure 2A,B)). Moreover, while the body weight
Nutrients 2024,16, 3138 6 of 30
change between yoyo RS- and HF RS-fed male mice did not differ (Tukey’s HSD: p> 0.9999
(Figure 2A)), the body weight change of the yoyo RS group was significantly lower than
HF RS-fed female mice (Tukey’s HSD: p= 0.0110) (Figure 2B).
Nutrients 2024, 16, x FOR PEER REVIEW 6 of 31
At the end of the diet intervention, regardless of sex, yoyo mice (green) had a similar
body weight change to control mice (blue) (two-way ANOVA diet × RS status in both sexes
p < 0.0001; Tukey’s HSD: male p = 0.8110 (Figure 2A); female p = 0.9995 (Figure 2B)) and
were significantly lighter than HF mice (red) (Tukey’s HSD: male p < 0.0001, female p =
0.0026 (Figure 2)).
Figure 2. Body weight change in male (A) and female mice (B). Weight change of mice fed control
(blue), HF (red), yoyo (green), control RS (purple), HF RS (yellow), and yoyo RS (black) diets. RS
supplemented in an HF diet (HF RS) significantly lowered body weight change in male mice com-
pared to an HF diet alone, but no significant difference was observed in female mice. N = 7–
8/group/sex; statistic at week 20 p ≤ 0.05 versus a control, b control RS, c HF RS, d yoyo, and e yoyo RS
mice. HF—high fat; RS—resistant starch.
Supplementation with RS in a control diet (control RS, purple) did not alter weight
gain in male or female mice in comparison with a control diet only (Tukey’s HSD: male p
> 0.9999 (Figure 2A), female p = 0.9994 (Figure 2B)). In contrast, RS supplementation in an
HF diet (HF RS) (yellow) resulted in significantly less weight gain than an HF diet alone
in male (Tukey’s HSD: p = 0.0002 (Figure 2A)) but not in female mice (Tukey’s HSD: p =
0.9995 (Figure 2B)).
With RS supplementation in the second phase of the yoyo diet (yoyo RS) (black),
there was no significant difference in the final body weight change compared to the yoyo
group (Tukey’s HSD: both sexes p > 0.9999 (Figure 2A,B)). Moreover, while the body
weight change between yoyo RS- and HF RS-fed male mice did not differ (Tukey’s HSD:
p > 0.9999 (Figure 2A)), the body weight change of the yoyo RS group was significantly
lower than HF RS-fed female mice (Tukey’s HSD: p = 0.0110) (Figure 2B).
3.1.2. Rates of Body Weight Gain during High-Fat Feeding Periods
Further analysis of body weight gain was performed on the HF and yoyo groups sup-
plemented with and without RS to explore the rate of weight regain of yoyo mice during
the two HF feeding periods: Phase 1 (weeks 0–5) and Phase 2 (weeks 10–15) (Figure 3).
Across the two HF feeding phases, male yoyo mice had a significantly higher rate of
weight gain compared to HF mice (+0.7171 g/week, p = 0.0087). However, in female mice,
HF and yoyo mice had similar rates of weight gain (p = 0.2158) (Figure 3).
Figure 2. Body weight change in male (A) and female mice (B). Weight change of mice fed control (blue),
HF (red), yoyo (green), control RS (purple), HF RS (yellow), and yoyo RS (black) diets. RS supplemented
in an HF diet (HF RS) significantly lowered body weight change in male mice compared to an HF diet
alone, but no significant difference was observed in female mice.
n= 7–8/group/sex
; statistic at week 20
p
≤
0.05 versus
a
control,
b
control RS,
c
HF RS,
d
yoyo, and
e
yoyo RS mice. HF—high fat; RS—resistant
starch.
3.1.2. Rates of Body Weight Gain during High-Fat Feeding Periods
Further analysis of body weight gain was performed on the HF and yoyo groups
supplemented with and without RS to explore the rate of weight regain of yoyo mice during
the two HF feeding periods: Phase 1 (weeks 0–5) and Phase 2 (weeks 10–15) (Figure 3).
Across the two HF feeding phases, male yoyo mice had a significantly higher rate of
weight gain compared to HF mice (+0.7171 g/week, p= 0.0087). However, in female mice,
HF and yoyo mice had similar rates of weight gain (p= 0.2158) (Figure 3).
Supplementation with RS in an HF diet (HF RS) resulted in a significantly reduced
rate of weight gain in male mice compared to those fed no RS (
−
1.28 g/week, p< 0.001). In
contrast, there were no significant differences in the rates of weight gain in female mice
(p= 0.79) (Figure 3).
With RS supplementation in the second phase of the yoyo diet (yoyo RS), male yoyo
RS mice gained significantly less weight compared to yoyo mice (
−
1.28 g/week, p< 0.0001).
However, the rate of weight gain of yoyo RS mice was still significantly higher than HF
RS mice (+1.44 g/week, p< 0.001) (Figure 3). Conversely, in female mice, there was no
significant difference in the rates of weight gain between the yoyo and yoyo RS groups
(p= 0.631) and between the yoyo RS and HF RS groups (p= 0.6405) (Figure 3).
Nutrients 2024,16, 3138 7 of 30
Nutrients 2024, 16, x FOR PEER REVIEW 7 of 31
Supplementation with RS in an HF diet (HF RS) resulted in a significantly reduced
rate of weight gain in male mice compared to those fed no RS (−1.28 g/week, p < 0.001). In
contrast, there were no significant differences in the rates of weight gain in female mice (p
= 0.79) (Figure 3).
With RS supplementation in the second phase of the yoyo diet (yoyo RS), male yoyo
RS mice gained significantly less weight compared to yoyo mice (−1.28 g/week, p < 0.0001).
However, the rate of weight gain of yoyo RS mice was still significantly higher than HF
RS mice (+1.44 g/week, p < 0.001) (Figure 3). Conversely, in female mice, there was no
significant difference in the rates of weight gain between the yoyo and yoyo RS groups (p
= 0.631) and between the yoyo RS and HF RS groups (p = 0.6405) (Figure 3).
Figure 3. Rate of weight regain of male and female mice in high-fat and yoyo groups, supplemented
with and without resistant starch, during two non-restricted (high-fat) feeding periods: Phase 1
(weeks 0–5) and Phase 2 (10–15). The weight of each animal is represented by an individual coloured
line. The difference in the rate of weight gain between the 2 phases was identified as the difference
in the average slopes of the same-coloured lines in Phase 1 and Phase 2. Bold blue solid lines indicate
the average rate of weight gain/loss in Phase 1 and Phase 2 per diet group, respectively. In male
mice, across the two HF feeding phases, yoyo mice had a significantly greater rate of weight gain
compared to HF mice. RS supplementation in HF and yoyo diets (HF RS and yoyo RS) resulted in
significantly lower rates of weight gain compared to diets supplemented with no RS. In female mice,
across the two HF feeding phases, HF and yoyo mice had similar rates of weight gain. RS supple-
mentation did not affect the rate of weight gain in female mice. HF—high-fat diet; RS—resistant
starch.
3.1.3. Fat Mass
Figure 3. Rate of weight regain of male and female mice in high-fat and yoyo groups, supplemented
with and without resistant starch, during two non-restricted (high-fat) feeding periods: Phase 1
(weeks 0–5) and Phase 2 (10–15). The weight of each animal is represented by an individual coloured
line. The difference in the rate of weight gain between the 2 phases was identified as the difference in
the average slopes of the same-coloured lines in Phase 1 and Phase 2. Bold blue solid lines indicate
the average rate of weight gain/loss in Phase 1 and Phase 2 per diet group, respectively. In male mice,
across the two HF feeding phases, yoyo mice had a significantly greater rate of weight gain compared
to HF mice. RS supplementation in HF and yoyo diets (HF RS and yoyo RS) resulted in significantly
lower rates of weight gain compared to diets supplemented with no RS. In female mice, across the
two HF feeding phases, HF and yoyo mice had similar rates of weight gain. RS supplementation did
not affect the rate of weight gain in female mice. HF—high-fat diet; RS—resistant starch.
3.1.3. Fat Mass
After 20 weeks, regardless of sex and RS status, yoyo mice (green) had similar gonadal
and perirenal fat mass compared to control mice (blue) (two-way ANOVA diet
×
RS status:
male p= 0.0232 and female p< 0.0001; Tukey’s HSD: male p= 0.8784 (Figure 4A) and
p= 0.9858 (Figure 4B); female p= 0.9816 (Figure 4A) and p= 0.8352 (Figure 4B), respectively)
and were significantly lower than HF mice (red) (Tukey’s HSD: male p= 0.0002 (Figure 4A)
and p< 0.0001 (Figure 4B), female p< 0.0001 (Figure 4A) and p= 0.0173 (Figure 4B),
respectively).
Supplementation with RS in mice fed a control diet (control RS) (purple) did not alter
gonadal and perirenal fat mass compared to those fed a control diet only (Tukey’s HSD:
Nutrients 2024,16, 3138 8 of 30
male p= 0.9991 (Figure 4A) and p= 0.8336 (Figure 4B), female p> 0.9999 (Figure 4A) and
p= 0.9716 (Figure 4B), respectively). In contrast, the supplementation of an HF diet with
RS (HF RS) (yellow) resulted in significantly less gonadal and perirenal fat mass than an
HF diet alone in male (Tukey’s HSD: p= 0.0085 (Figure 4A) and p< 0.0001 (Figure 4B),
respectively) but not in female mice (Tukey’s HSD: p> 0.9999 (Figure 4A) and p= 0.9849
(Figure 4B), respectively).
With RS supplementation in the second phase of the yoyo diet (yoyo RS) (black),
there was no significant difference in gonadal and perirenal fat mass compared to the
yoyo group (Tukey’s HSD: male p> 0.9999 (Figure 4A) and p= 0.9978 (Figure 4B), female
p> 0.9999 (Figure 4A) and p= 0.8847 (Figure 4B), respectively). Gonadal and perirenal
fat mass between male yoyo RS mice and male HF RS mice did not differ (Tukey’s HSD:
p= 0.8332 (Figure 4A) and p= 0.9469 (Figure 4B), respectively). However, gonadal fat
mass in female yoyo RS mice was significantly lower than in HF RS mice (Tukey’s HSD:
p< 0.0001 (Figure 4A).
Nutrients 2024, 16, x FOR PEER REVIEW 8 of 31
After 20 weeks, regardless of sex and RS status, yoyo mice (green) had similar gon-
adal and perirenal fat mass compared to control mice (blue) (two-way ANOVA diet × RS
status: male p = 0.0232 and female p < 0.0001; Tukey’s HSD: male p = 0.8784 (Figure 4A)
and p = 0.9858 (Figure 4B); female p = 0.9816 (Figure 4A) and p = 0.8352 (Figure 4B), respec-
tively) and were significantly lower than HF mice (red) (Tukey’s HSD: male p = 0.0002
(Figure 4A) and p < 0.0001 (Figure 4B), female p < 0.0001 (Figure 4A) and p = 0.0173 (Figure
4B), respectively).
Supplementation with RS in mice fed a control diet (control RS) (purple) did not alter
gonadal and perirenal fat mass compared to those fed a control diet only (Tukey’s HSD:
male p = 0.9991 (Figure 4A) and p = 0.8336 (Figure 4B), female p > 0.9999 (Figure 4A) and p
= 0.9716 (Figure 4B), respectively). In contrast, the supplementation of an HF diet with RS
(HF RS) (yellow) resulted in significantly less gonadal and perirenal fat mass than an HF
diet alone in male (Tukey’s HSD: p = 0.0085 (Figure 4A) and p < 0.0001 (Figure 4B), respec-
tively) but not in female mice (Tukey’s HSD: p > 0.9999 (Figure 4A) and p = 0.9849 (Figure
4B), respectively).
With RS supplementation in the second phase of the yoyo diet (yoyo RS) (black),
there was no significant difference in gonadal and perirenal fat mass compared to the yoyo
group (Tukey’s HSD: male p > 0.9999 (Figure 4A) and p = 0.9978 (Figure 4B), female p >
0.9999 (Figure 4A) and p = 0.8847 (Figure 4B), respectively). Gonadal and perirenal fat mass
between male yoyo RS mice and male HF RS mice did not differ (Tukey’s HSD: p = 0.8332
(Figure 4A) and p = 0.9469 (Figure 4B), respectively). However, gonadal fat mass in female
yoyo RS mice was significantly lower than in HF RS mice (Tukey’s HSD: p < 0.0001 (Figure
4A).
Figure 4. Fat mass. Gonadal fat (A). Perirenal fat (B). RS supplemented in an HF diet (HF RS)
resulted in a significant reduction in fat mass in male mice compared to an HF diet alone, but no
significant difference was observed in female mice. * p
≤
0.05, *** p
≤
0.001, **** p
≤
0.0001, with
n= 7–8/group/sex.
3.2. Liver Health
At the end of the dietary intervention, yoyo dieting improved indices of liver health
in male mice compared to HF feeding alone. In particular, male yoyo mice (green) had
similar a liver mass, liver triglycerides (two-way ANOVA diet
×
RS status, both p< 0.0001;
Tukey’s HSD: both p> 0.9999 (Figure 5A,B)), and hepatocyte ballooning (two-way ANOVA
Nutrients 2024,16, 3138 9 of 30
diet
×
RS status, p= 0.0009; Tukey’s HSD: p= 0.8740 (Figure 5C,E,I)) compared to control
mice (blue). Additionally, male yoyo mice had a significantly lower liver mass and triglyc-
erides, but not hepatocyte ballooning, compared to HF mice (red) (Tukey’s HSD: p= 0.0003
(Figure 5A), p= 0.5401 (Figure 5B), and p= 0.6313 (Figure 5I), respectively). Conversely,
regardless of diet and RS status, there were no differences in liver mass (two-way ANOVA
diet
×
RS status, p= 0.3927 (Figure 5A)), liver triglycerides (two-way ANOVA diet
×
RS
status, p= 0.9606 (Figure 5B)), or hepatocyte ballooning (two-way ANOVA diet
×
RS status,
p= 0.7146 (Figure 5I)) in female mice.
Supplementation with RS in male mice fed a control diet (control RS) (purple) did not
alter the liver mass (Tukey’s HSD: p= 0.9001 (Figure 5A)), liver triglycerides (Tukey’s HSD:
p> 0.9999 (Figure 5B)), or hepatocyte ballooning (Tukey’s HSD: p= 0.6431 (Figure 5C,F,I))
compared to a control diet alone. However, supplementation with RS in an HF diet (HF RS)
(yellow) resulted in a significantly lower liver mass (Tukey’s HSD: p= 0.0001 (Figure 5A)),
liver triglycerides (Tukey’s HSD: p< 0.0001 (Figure 5B)), and hepatocyte ballooning (Tukey’s
HSD: p= 0.0109 (Figure 5C,D,I)) compared to an HF diet alone.
With RS supplementation in the second phase of the yoyo diet (yoyo RS) (black), there
were no significant differences in liver mass, liver triglycerides, and hepatocyte ballooning
compared to the yoyo group (Tukey’s HSD: p> 0.9999 (Figure 5A), p= 0.9994 (Figure 5B),
and p= 0.2437 (Figure 5E,H,I), respectively) and between the yoyo RS and HF RS groups
(Tukey’s HSD: p= 0.9999 (Figure 5A), p> 0.9999 (Figure 5B), and p= 0.5663 (Figure 5G–I),
respectively).
3.3. Blood Glucose Metabolism
In male mice, there was no significant difference in glucose and insulin levels during
an OGTT, and glucose levels during an ITT between the HF (red) and control (blue) groups
(two-way ANOVA diet
×
RS status, p= 0.0664 (Figure 6A), p= 0.1893 (Figure 6B), p= 0.0004
(Figure 6C); Tukey’s HSD: p= 0.9545, p= 0.1468, p= 0.9702, respectively) and between the
yoyo and control groups (Tukey’s HSD: p= 0.7229, p= 0.8876, p= 0.7367, respectively).
Yoyo dieting (green) appeared to improve blood glucose homeostasis compared to contin-
uous HF feeding; however, this was not significantly different (Tukey’s HSD: p= 0.2536
(Figure 6A), p= 0.6849 (Figure 6B), p= 0.3299 (Figure 6C), respectively). In female mice,
HF mice had significantly higher glucose tolerance (two-way ANOVA diet
×
RS status,
p< 0.0001; Tukey’s HSD: p= 0.0006 (Figure 6A)) but similar insulin levels during an OGTT
(two-way ANOVA diet
×
RS status, p= 0.004; Tukey’s HSD: p= 0.1906 (Figure 6B)) and
glucose levels during an ITT (two-way ANOVA diet
×
RS status, p= 0.0234; Tukey’s HSD:
p= 0.0502 (Figure 6C)) compared to control mice. There was no significant difference in glu-
cose homeostasis and plasma insulin between the yoyo and control groups (Tukey’s HSD:
p= 0.1312 (Figure 6A), p> 0.9999 (Figure 6B), p= 0.8399 (Figure 6C), respectively). Yoyo
dieting significantly lowered glucose and insulin levels during an OGTT, but not ITT,
compared to HF mice (Tukey’s HSD: p< 0.0001 (Figure 6A), p= 0.0286 (Figure 6B),
p= 0.2294 (Figure 6C), respectively).
Supplementation with RS in mice fed a control diet (control RS) (purple) did not
significantly alter glucose and insulin measured during an OGTT or insulin tolerance
compared to mice fed a control diet only (male Tukey’s HSD: p= 0.7583 (Figure 6A),
p= 0.8321 (Figure 6B), p= 0.7941 (Figure 6C), respectively; female Tukey’s HSD: p= 0.8731
(Figure 6A), p= 0.8374 (Figure 6B), p= 0.0528 (Figure 6C), respectively). Similarly, RS
supplementation in mice fed an HF diet (HF RS) (yellow) did not significantly alter glucose
and insulin levels during an OGTT compared to HF mice (male Tukey’s HSD: p= 0.2760
(Figure 6A), p= 0.9685 (Figure 6B), respectively; female Tukey’s HSD: p> 0.9999 (Figure 6A),
p= 0.6550 (Figure 6B), respectively). However, male mice consuming an HF RS diet had
significantly improved insulin tolerance compared to mice fed an HF diet alone (Tukey’s
HSD: p= 0.0123 (Figure 6C)).
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Figure 5. Indices of liver health. Liver mass (A). Liver triglycerides (B). Hepatocyte ballooning im-
ages of male mice using haematoxylin and eosin staining (C–H). Hepatocyte ballooning (I). RS sup-
plementation in an HF diet (HF RS) significantly improved the liver health of male mice compared
Figure 5. Indices of liver health. Liver mass (A). Liver triglycerides (B). Hepatocyte ballooning
images of male mice using haematoxylin and eosin staining (C–H). Hepatocyte ballooning (I). RS
supplementation in an HF diet (HF RS) significantly improved the liver health of male mice compared
to an HF diet only, but no significant difference was observed in female mice. * p
≤
0.05, *** p
≤
0.001,
**** p≤0.0001, n= 7–8/group/sex. Scale bar = 150 µm. RS: resistant starch.
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to an HF diet only, but no significant difference was observed in female mice. * p ≤ 0.05, *** p ≤ 0.001,
**** p ≤ 0.0001, n = 7–8/group/sex. Scale bar = 150 µm. RS: resistant starch.
Supplementation with RS in mice fed a control diet (control RS) (purple) did not sig-
nificantly alter glucose and insulin measured during an OGTT or insulin tolerance com-
pared to mice fed a control diet only (male Tukey’s HSD: p = 0.7583 (Figure 6A), p = 0.8321
(Figure 6B), p = 0.7941 (Figure 6C), respectively; female Tukey’s HSD: p = 0.8731 (Figure
6A), p = 0.8374 (Figure 6B), p = 0.0528 (Figure 6C), respectively). Similarly, RS supplemen-
tation in mice fed an HF diet (HF RS) (yellow) did not significantly alter glucose and in-
sulin levels during an OGTT compared to HF mice (male Tukey’s HSD: p = 0.2760 (Figure
6A), p = 0.9685 (Figure 6B), respectively; female Tukey’s HSD: p > 0.9999 (Figure 6A), p =
0.6550 (Figure 6B), respectively). However, male mice consuming an HF RS diet had sig-
nificantly improved insulin tolerance compared to mice fed an HF diet alone (Tukey’s
HSD: p = 0.0123 (Figure 6C)).
With RS supplementation in the second phase of the yoyo diet (yoyo RS) (black),
there were no significant differences in glucose and insulin levels during an OGTT or in
glucose during an ITT between yoyo RS and yoyo mice (male Tukey’s HSD: p = 0.9852
(Figure 6A), p = 0.9909 (Figure 6B), p = 0.7946 (Figure 6C), respectively; female Tukey’s
HSD: p = 0.5349 (Figure 6A), p > 0.9999 (Figure 6B), p = 0.9992 (Figure 6C), respectively).
Similarly, there was no difference in glucose and insulin levels in male yoyo RS and HF
RS mice (Tukey’s HSD: p = 0.9892 (Figure 6A), p > 0.9999 (Figure 6B), p = 0.8179 (Figure
6C), respectively). On the other hand, female yoyo RS mice had significantly improved
glucose tolerance compared to HF RS mice (Tukey’s HSD: p < 0.0001 (Figure 6A)).
Figure 6. Metabolic measurements. Blood glucose during an oral glucose tolerance test (A). Plasma
insulin during an oral glucose tolerance test (B). Blood glucose during an insulin tolerance test (C).
Figure 6. Metabolic measurements. Blood glucose during an oral glucose tolerance test (A). Plasma
insulin during an oral glucose tolerance test (B). Blood glucose during an insulin tolerance test (C).
RS supplementation in an HF diet (HF RS) resulted in a significantly improved insulin tolerance
in male mice compared to an HF diet alone, but no significant difference was observed in female
mice. * p
≤
0.05, ** p
≤
0.01, *** p
≤
0.001, and **** p
≤
0.0001 used to determine significance, with
n= 7–8/group/sex. AUC: area under the curve.
With RS supplementation in the second phase of the yoyo diet (yoyo RS) (black), there
were no significant differences in glucose and insulin levels during an OGTT or in glucose
during an ITT between yoyo RS and yoyo mice (male Tukey’s HSD: p= 0.9852 (Figure 6A),
p= 0.9909 (Figure 6B), p= 0.7946 (Figure 6C), respectively; female Tukey’s HSD: p= 0.5349
(Figure 6A), p> 0.9999 (Figure 6B), p= 0.9992 (Figure 6C), respectively). Similarly, there was
no difference in glucose and insulin levels in male yoyo RS and HF RS mice (Tukey’s HSD:
p= 0.9892 (Figure 6A), p> 0.9999 (Figure 6B), p= 0.8179 (Figure 6C), respectively). On the
other hand, female yoyo RS mice had significantly improved glucose tolerance compared
to HF RS mice (Tukey’s HSD: p< 0.0001 (Figure 6A)).
3.4. Short-Chain Fatty Acid Concentration
There were no significant differences in SCFA levels across all diets (propionate: two-
way ANOVA diet
×
RS status male p= 0.3066, female p= 0.0705; n–Butyrate: two-way
ANOVA diet ×RS status male p= 0.1932, female p= 0.4170) (Figure 7).
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RS supplementation in an HF diet (HF RS) resulted in a significantly improved insulin tolerance in
male mice compared to an HF diet alone, but no significant difference was observed in female mice.
* p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, and **** p ≤ 0.0001 used to determine significance, with n = 7–
8/group/sex. AUC: area under the curve.
3.4. Short-Chain Fay Acid Concentration
There were no significant differences in SCFA levels across all diets (propionate: two-
way ANOVA diet × RS status male p = 0.3066, female p = 0.0705; n–Butyrate: two-way
ANOVA diet × RS status male p = 0.1932, female p = 0.4170) (Figure 7).
Figure 7. Short-chain fay acid levels. n–Butyrate (A). Propionate (B). No change in SCFA levels
across diet groups. n = 7–8/group/sex.
3.5. Resistant Starch and Yoyo Dieting Reshaped Gut Microbiota
3.5.1. Relative Abundance by Phylum
From a simple visual inspection, in male mice, during the second phase of HF feed-
ing, yoyo mice had enriched Bacteroidetes (23.2%) compared to HF (17.7%), but it was
reduced compared to control mice (37.6%). However, the relative abundance of Firmicutes
in yoyo mice (74.3%) was reduced compared to HF mice (79.7%) but enriched compared
to controls (55.4%). By the end of the diet intervention, yoyo mice had lower Bacteroidetes
(40.8%) and similar Firmicutes (48.6%) compared to controls (44.0% and 47.8%, respec-
tively), but they had enriched Bacteroidetes and lower Firmicutes compared to HF mice
(32.9% and 62.5%, respectively) (Figure 8, male, week 15). In female mice, during the sec-
ond phase of HF feeding, yoyo mice had a lower relative abundance of Bacteroidetes
(44.2%) than HF mice (49%), but it was higher than in control mice (29.1%). However, the
Figure 7. Short-chain fatty acid levels. n-Butyrate (A). Propionate (B). No change in SCFA levels
across diet groups. n= 7–8/group/sex.
3.5. Resistant Starch and Yoyo Dieting Reshaped Gut Microbiota
3.5.1. Relative Abundance by Phylum
From a simple visual inspection, in male mice, during the second phase of HF feeding,
yoyo mice had enriched Bacteroidetes (23.2%) compared to HF (17.7%), but it was reduced
compared to control mice (37.6%). However, the relative abundance of Firmicutes in yoyo
mice (74.3%) was reduced compared to HF mice (79.7%) but enriched compared to controls
(55.4%). By the end of the diet intervention, yoyo mice had lower Bacteroidetes (40.8%) and
similar Firmicutes (48.6%) compared to controls (44.0% and 47.8%, respectively), but they
had enriched Bacteroidetes and lower Firmicutes compared to HF mice (32.9% and 62.5%,
respectively) (Figure 8, male, week 15). In female mice, during the second phase of HF
feeding, yoyo mice had a lower relative abundance of Bacteroidetes (44.2%) than HF mice
(49%), but it was higher than in control mice (29.1%). However, the relative abundance of
Firmicutes between yoyo (52.0%), HF (49.3%), and control mice (53.2%) did not differ. By
the end of the diet intervention, yoyo mice had lower Bacteroidetes (30.7%) and enriched
Firmicutes (56.3%) compared to HF (56.8% and 39.4%, respectively) and control mice (37.0%
and 45.2%, respectively) (Figure 8, female, week 15).
Supplementation with RS in a control diet (control RS) was associated with a greater
relative abundance of Actinobacteria compared to a control diet only (Figure 8, male, week
15: 21.8% versus 0.63%, week 20: 19.09% versus 1.6%; Figure 8, female, week 15: 12.9%
versus 0.05%, week 20: 7.73% versus 0.09%). Likewise, RS supplementation in an HF diet
(HF RS) was also associated with enriched Actinobacteria compared to an HF diet only
(Figure 8, male, week 15: 33.9% versus 0.26%, week 20: 34.9% versus 1.5%; Figure 8, female,
week 15: 13.08% versus 0.15%, week 20: 3.46% versus 0.21%).
Nutrients 2024,16, 3138 13 of 30
During the second phase of HF feeding, RS supplementation in the yoyo diet (yoyo
RS) resulted in a greater relative abundance of Actinobacteria compared to the yoyo
diet supplemented without RS (Figure 8, male, week 15: 59.3% versus 0.1%; Figure 8,
female, week 15: 15.4% versus 7.36%, respectively). Similarly, yoyo RS mice had increased
Actinobacteria compared to HF RS mice (Figure 8, male, week 15: 59.3% versus 33.9%,
Figure 8, female, week 15: 15.4% versus 13.08%) (Figure 8).
Nutrients 2024, 16, x FOR PEER REVIEW 13 of 31
relative abundance of Firmicutes between yoyo (52.0%), HF (49.3%), and control mice
(53.2%) did not differ. By the end of the diet intervention, yoyo mice had lower Bacteroide-
tes (30.7%) and enriched Firmicutes (56.3%) compared to HF (56.8% and 39.4%, respec-
tively) and control mice (37.0% and 45.2%, respectively) (Figure 8, female, week 15).
Supplementation with RS in a control diet (control RS) was associated with a greater
relative abundance of Actinobacteria compared to a control diet only (Figure 8, male, week
15: 21.8% versus 0.63%, week 20: 19.09% versus 1.6%; Figure 8, female, week 15: 12.9%
versus 0.05%, week 20: 7.73% versus 0.09%). Likewise, RS supplementation in an HF diet
(HF RS) was also associated with enriched Actinobacteria compared to an HF diet only
(Figure 8, male, week 15: 33.9% versus 0.26%, week 20: 34.9% versus 1.5%; Figure 8, fe-
male, week 15: 13.08% versus 0.15%, week 20: 3.46% versus 0.21%).
During the second phase of HF feeding, RS supplementation in the yoyo diet (yoyo
RS) resulted in a greater relative abundance of Actinobacteria compared to the yoyo diet
supplemented without RS (Figure 8, male, week 15: 59.3% versus 0.1%; Figure 8, female,
week 15: 15.4% versus 7.36%, respectively). Similarly, yoyo RS mice had increased Actino-
bacteria compared to HF RS mice (Figure 8, male, week 15: 59.3% versus 33.9%, Figure 8,
female, week 15: 15.4% versus 13.08%) (Figure 8).
Figure 8. Relative abundance. Stacked column charts representing the relative abundance of am-
plicon sequence variants (ASVs) at the taxonomic level of the phylum. The visualisation of ASV data
displays the gut microbiota of male and female mice consuming 6 different diets in weeks 15 and
20. Diets supplemented with RS were associated with a higher relative abundance of Actinobacteria.
HF: high fat. RS: resistant starch.
3.5.2. Alpha Diversity
Alpha diversity indices are used to describe the microbial diversity within an ecolog-
ical community with respect to its richness and/or evenness [102]. The Fisher index con-
siders ASV richness, and the Shannon index considers both richness and evenness.
Figure 8. Relative abundance. Stacked column charts representing the relative abundance of amplicon
sequence variants (ASVs) at the taxonomic level of the phylum. The visualisation of ASV data displays
the gut microbiota of male and female mice consuming 6 different diets in weeks 15 and 20. Diets
supplemented with RS were associated with a higher relative abundance of Actinobacteria. HF: high
fat. RS: resistant starch.
3.5.2. Alpha Diversity
Alpha diversity indices are used to describe the microbial diversity within an ecological
community with respect to its richness and/or evenness [
102
]. The Fisher index considers
ASV richness, and the Shannon index considers both richness and evenness.
At the end of the diet intervention, male mice fed an HF diet (red) had significantly
lower alpha diversity than control mice (blue) (Fisher p= 0.015, Shannon p= 0.001). Male
yoyo mice (green) appeared to have an alpha diversity that was in an intermediate state
between control and HF mice; however, this was not significantly different (yoyo versus
control: Fisher p= 0.70 and Shannon p= 0.97; yoyo versus HF: Fisher p= 0.16 and Shannon
p= 0.07 (Figure 9A, week 20)). In female mice, there were no significant differences in
alpha diversity at the end of the diet intervention between the HF, yoyo, and control groups
(Fisher and Shannon—yoyo versus HF: p= 0.36 and p= 0.07; yoyo versus control: p= 0.23
and p= 0.31; HF versus control: p= 0.91 and p= 0.73, respectively (Figure 9B, week 20)).
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Figure 9. Alpha diversity. Fisher’s and Shannon’s diversity indices of faecal samples of male (A) and
female mice (B) in week 15 and week 20. The median is illustrated by the horizontal line inside the
box. The lowest and highest values within 1.5 times the interquartile range from the 1st and 3rd
quartiles, respectively, are illustrated by whiskers. * p ≤ 0.05, ** p ≤ 0.01, with n = 6–8/group/sex.
Boxes represent the interquartile range between the first and third quartiles. The horizontal line
inside the box illustrates the median. Solid dots (●) outside the whiskers indicate greater than 1.5
times and less than 3 times the interquartile range. Graphs were generated from raw and untrimmed
data. HF—high-fat diet; RS—resistant starch.
Figure 9. Alpha diversity. Fisher ’s and Shannon’s diversity indices of faecal samples of male (A)
and female mice (B) in week 15 and week 20. The median is illustrated by the horizontal line inside
the box. The lowest and highest values within 1.5 times the interquartile range from the 1st and 3rd
quartiles, respectively, are illustrated by whiskers. * p
≤
0.05, ** p
≤
0.01, with n= 6–8/group/sex.
Boxes represent the interquartile range between the first and third quartiles. The horizontal line inside
the box illustrates the median. Solid dots (
•
) outside the whiskers indicate greater than 1.5 times
and less than 3 times the interquartile range. Graphs were generated from raw and untrimmed data.
HF—high-fat diet; RS—resistant starch.
Additionally, supplementation with RS in a control diet (control RS) (purple) sig-
nificantly reduced alpha diversity in comparison with a control diet only (male: Fisher
p= 0.005, Shannon p= 0.001 (Figure 9A, week 20); female: Fisher p= 0.11, Shannon
p= 0.04 (Figure 9B, week 20)). Similarly, RS supplementation in an HF diet (HF RS) (yellow)
Nutrients 2024,16, 3138 15 of 30
reduced alpha diversity compared to an HF diet alone in male (Fisher p= 0.05, Shannon
p= 0.04 (Figure 9A, week 20)) but not in female mice (Fisher p= 0.76, Shannon p= 0.97
(Figure 9B, week 20)).
Likewise, RS supplementation in the second phase of the yoyo diet (yoyo RS) (black)
resulted in significantly reduced alpha diversity compared to the yoyo group in male
(Fisher p= 0.015, Shannon p= 0.007 (Figure 9A, week 15)) but not in female mice (Fisher
p= 0.91, Shannon p= 0.97 (Figure 9B, week 15)).
3.5.3. Beta Diversity
The Bray–Curtis measure was used to describe similarities and dissimilarities between
diet treatment groups based on their gut microbial community members and abundances,
and a principal coordinate analysis (PCoA) plot was constructed (Figure 10).
Nutrients 2024, 16, x FOR PEER REVIEW 16 of 31
Figure 10. Principal coordinate analysis (PCoA)—beta diversity. Bray–Curtis ordination plot show-
ing the dissimilarity of the gut microbiota from mice fed six different diets at two time points (weeks
15 and 20). The microbiota profiles of different dietary treatments are represented by different col-
ours: blue (control), purple (control RS), red (HF), yellow (HF RS), green (yoyo), and black (yoyo
RS). Solid arrows represent female mice, and arrows with dashed lines represent male mice. The tail
of an arrow indicates microbiota composition in week 15, while the tip of an arrow indicates micro-
biota composition in week 20. RS—resistant starch; HF—high fat.
3.5.4. Differential Abundance Analysis
At the end of the diet intervention, we observed 16 (8 male; 8 female) differentially
abundant genera between HF and control mice (Figures 11 and 12). HF-fed mice had sig-
nificantly enriched Clostridium (male: log2FC = 3.78; female: log2FC = 3.49) and reduced
Anaerotruncus (male: log2FC = 3.33; female: log2FC = 4.40) compared to control mice. On
the other hand, we observed 25 (12 male; 13 female) differentially abundant genera be-
tween HF and yoyo mice. HF-fed mice had significantly reduced Anaerotruncus (male:
log2FC = –2.89; female: log2FC = –4.15), Bilophila (male: log2FC = –1.92; female: log2FC = –
2.05), and Coprococcus (male: log2FC = –1.38; female: log2FC = –2.07) compared to yoyo
mice. Additionally, there were six (two male; four female) differentially abundant genera
between yoyo and control mice. Male yoyo mice had significantly reduced Prevotella (see
Supplementary Material for details on this classification) (log2FC = –25.8) and Lactobacillus
(log2FC = –3.1) (Figure 11), while female yoyo mice had significantly reduced Anaero-
plasma (log2FC = –20.9) and Bacteroides (log2FC = –3.4), but enriched Clostridium (log2FC =
3.7) and Bifidobacterium (log2FC = 12.8), compared to control mice (Figure 12).
Figure 10. Principal coordinate analysis (PCoA)—beta diversity. Bray–Curtis ordination plot showing
the dissimilarity of the gut microbiota from mice fed six different diets at two time points (weeks 15
and 20). The microbiota profiles of different dietary treatments are represented by different colours:
blue (control), purple (control RS), red (HF), yellow (HF RS), green (yoyo), and black (yoyo RS). Solid
arrows represent female mice, and arrows with dashed lines represent male mice. The tail of an
arrow indicates microbiota composition in week 15, while the tip of an arrow indicates microbiota
composition in week 20. RS—resistant starch; HF—high fat.
HF mice (red) had a significantly different microbiota composition compared to control
mice (blue) (adjusted for sex, R2= 0.13, p= 0.001). Supplementation with RS regardless of
diet (purple, yellow, black) also resulted in significantly different microbiota compositions
compared to those supplemented without RS (blue, red, green) (adjusted for sex, R
2
= 0.0650,
p= 0.001). Moreover, we observed significantly different gut microbiota compositions in
male and female mice (R2= 0.0575, p= 0.001) (Figure 10).
Nutrients 2024,16, 3138 16 of 30
At the end of the second HF feeding phase, the microbiota signatures of yoyo mice were
significantly different from the control (week 15, adjusted for sex, R
2
= 0.22, p= 0.001) and HF
states (week 15: adjusted for sex, R
2
= 0.05, p= 0.045). Moreover, by the end of the dietary
intervention, this significantly altered microbiota composition of yoyo mice persisted even
after a 5-week control feeding period and achieving weight loss (week 20: yoyo versus control:
adjusted for sex, R
2
= 0.09, p= 0.002; yoyo versus HF: adjusted for sex, R
2
= 0.17, p= 0.001).
Interestingly, RS supplementation in the second phase of the yoyo diet in male mice (yoyo RS)
(black) appeared to exhibit a shift in the gut microbiota composition that was closer to the
control group by the end of the dietary intervention. However, the microbiota profile of male
yoyo RS mice remained significantly different from control mice (week 20, black versus blue,
R2= 0.26, p= 0.001). No such shift was observed in female yoyo RS mice (Figure 10).
3.5.4. Differential Abundance Analysis
At the end of the diet intervention, we observed 16 (8 male; 8 female) differentially
abundant genera between HF and control mice (Figures 11 and 12). HF-fed mice had
significantly enriched Clostridium (male: log2FC = 3.78; female: log2FC = 3.49) and re-
duced Anaerotruncus (male: log2FC = 3.33; female: log2FC = 4.40) compared to control
mice. On the other hand, we observed 25 (12 male; 13 female) differentially abundant
genera between HF and yoyo mice. HF-fed mice had significantly reduced Anaerotruncus
(male: log2FC =
−
2.89; female: log2FC =
−
4.15), Bilophila (male: log2FC =
−
1.92; female:
log2FC =
−
2.05), and Coprococcus (male: log2FC =
−
1.38; female: log2FC =
−
2.07) compared
to yoyo mice. Additionally, there were six (two male; four female) differentially abundant
genera between yoyo and control mice. Male yoyo mice had significantly reduced Prevotella
(see Supplementary Materials for details on this classification) (log2FC =
−
25.8) and Lac-
tobacillus (log2FC =
−
3.1) (Figure 11), while female yoyo mice had significantly reduced
Anaeroplasma (log2FC =
−
20.9) and Bacteroides (log2FC =
−
3.4), but enriched Clostridium
(log2FC = 3.7) and Bifidobacterium (log2FC = 12.8), compared to control mice (Figure 12).
Supplementation with RS in a control diet (control RS) resulted in 16 (5 male;
11 female) differentially abundant genera in comparison with a control diet only, par-
ticularly markedly increased Parabacteroides (male: log2FC = 4.31; female: log2FC = 3.16),
Bifidobacterium (male: log2FC = 5.03 [female: log2FC = 16.16), and Desulfovibrio (male:
log2FC = 9.32; female: log2FC = 6.31). Similarly, RS supplementation in an HF diet (HF
RS) resulted in 30 (19 male; 11 female) differentially abundant genera compared to an HF
diet alone, particularly enriched Parabacteroides (male: log2FC = 3.46; female: log2FC = 2.99)
and Bifidobacterium (male: log2FC = 6.16; female: log2FC = 5.47) (Figures 11 and 12).
In males, RS supplementation in the second phase of the yoyo diet (yoyo RS) re-
sulted in 15 differentially abundant genera (Figures 11 and 12). In particular, yoyo RS
mice had enriched Parabacteroides (log2FC = 5.08), Bifidobacterium (log2FC = 12.35), Desul-
fovibrio (log2FC = 9.46), Lactobacillus (log2FC = 5.09), and Adlercreutzia (log2FC = 3.71)
but reduced Ruminococcus (log2FC =
−
1.12) compared to yoyo mice. Interestingly, at the
end of the dietary intervention, we only observed three differentially abundant genera in
yoyo RS mice compared to yoyo mice, including Prevotella (log2FC = 26.63), Anaeroplasma
(log2FC =
−
22.18), and Adlercreutzia log2FC = 4.02) (Figure 11). On the other hand, in
female mice, we observed 11 differentially abundant genera between yoyo RS and yoyo
mice. Particularly, yoyo RS mice had significantly enriched Bifidobacterium (log2FC = 6.15),
Parabacteroides (log2FC = 5.33), and Bacteroides (log2FC = 3.31) but reduced Desulfovibrio
(log2FC = −6.32) and Dorea (log2FC = −2.93) compared to yoyo mice (Figure 12).
In order to explore which genera may have accounted for the shift in beta diversity
closer to the control state of male yoyo RS mice, we observed nine differentially abundant
genera. At the end of the dietary intervention, compared to the second phase of HF feeding
with RS supplementation, yoyo RS mice exhibited significantly enriched Ruminococcus (also
known as Mediterraneibacter, refer to Supplementary Materials for more details)
(log2FC = 1.67)
and significantly reduced Parabacteroides (log2FC =
−
3.90), Bifidobacterium
(log2FC = −6.79)
,
Desulfovibrio (log2FC = −9.64), and Lactobacillus (log2FC = −4.74) (Figure 11).
Nutrients 2024,16, 3138 17 of 30
Nutrients 2024, 16, x FOR PEER REVIEW 17 of 31
Figure 11. Differentially abundant genera in male mice. Only genera that were significantly different
in relative abundance (FDR
≤
0.01) are shown in the plot, with log2FoldChange estimated by DESeq2.
RS—resistant starch; HF—high fat; W—week.
Nutrients 2024,16, 3138 18 of 30
Nutrients 2024, 16, x FOR PEER REVIEW 19 of 31
Figure 12. Differentially abundant genera in female mice. Only genera that were significantly
different in relative abundance (FDR ≤0.01) are shown in the plot, with log2FoldChange estimated
by DESeq2. RS—resistant starch; HF—high fat; W—week.
Nutrients 2024,16, 3138 19 of 30
4. Discussion
Relatively few studies have evaluated the effect of yoyo dieting and RS supplemen-
tation on metabolic and gut health. The present study revealed that yoyo dieting is both
beneficial and detrimental to metabolic health, with the gut microbiome composition
remaining in an intermediate state of dysbiosis. We also showed the beneficial effects
of RS supplementation in improving metabolic outcomes, in addition to reshaping the
gut microbiome.
4.1. Yoyo Dieting Appears to Be a Double-Edged Sword: Beneficial and Deleterious Effects of Yoyo
Dieting on Metabolic and Gut Health
While there have been controversial findings around the effects of yoyo dieting on the
metabolism, our study revealed similar metabolic outcomes following yoyo and control
dieting. This aligns with previous studies [
47
,
103
] highlighting the overall importance
of weight loss in improving metabolic health despite yoyo dieting. However, despite
the benefits of weight loss, we also observed that yoyo dieting led to a greater rate of
weight regain in male but not female mice, compared to continuous HF feeding, potentially
due to gut microbial differences between sexes. This is consistent with previous studies
suggesting that yoyo dieting may be a risk factor in future weight regain and obesity
development [
47
,
104
,
105
]. Given the sex differences that we observed in metabolic health
and gut microbiome changes, our study further emphasises the need to investigate both
sexes in metabolic studies given the paucity of studies in this area.
It is well established that HF diet feeding alters the gut microbiota composition
[55,106,107]
.
There is also increasing evidence to suggest that yoyo dieting results in significantly different
gut bacterial diversity and long–term alterations in gut microbiota composition
[47,48]
. A study
by Thaiss et al. [
47
] investigated the long–term effects of yoyo dieting on mice exposed to two
cycles of yoyo dieting. They found that despite achieving successful weight loss, the microbiota
profile of yoyo mice remained in an intermediate configuration between the obese and control
states. Furthermore, this post-dieting microbiome signature persisted and required a period
more than five times longer than the last dieting period to reverse back to the control state. Our
results are consistent with this study as we found different bacterial signatures and bacterial
diversity from the control and HF states, despite successful weight loss. Collectively, these
results imply that although the weight loss following yoyo dieting has short–term benefits on
metabolic health [
108
], it is possible that the intermediate gut microbiome signature present
in yoyo dieting may play a role in increasing the susceptibility to weight regain shortly after
weight loss.
4.2. Beneficial Effects of Resistant Starch on Metabolic Health
Our findings highlight the beneficial effects of RS in improving metabolic outcomes,
which are in line with numerous animal [
69
,
109
–
116
] and human studies [
53
,
117
–
121
].
Additionally, our study shows a striking protective effect of RS in improving metabolic
outcomes, particularly in reducing body weight in male HF RS mice only. It is also
important to note that there were no clinical signs of illness or changes in food intake in
any of our dietary groups. The observed weight loss with RS supplementation in the HF
diet (HF RS) during the second half of our study (particularly during weeks 11–16) may be
due to impaired nutrient absorption and/or increased energy expenditure, which we were
not able to measure in this study. Therefore, future studies are warranted to verify this
hypothesis. There are also inconsistencies in the literature around RS and weight loss that
could be attributed to several factors, such as variations in experimental models, treatment
durations, types of RS, and different treatment dosages. Moreover, most of what we know
about the effects of RS is based on animal and human studies that have mainly used
RS2 (raw potato, green banana, high-amylose maize starch)
[52,53,69,111,113,116,122–128]
,
with limited studies on RS4 (chemically modified starch) [
129
–
132
]. Although RS2 is
more commonly found in foods, it is less resistant and easily degraded by heat (such as
Nutrients 2024,16, 3138 20 of 30
in cooking). In contrast, RS4 offers significantly greater chemical resistance to digestive
enzymes in the gut compared to RS2 [80,133].
RS represents a small portion in most foods (<2.5%, dry matter basis), even in starchy
foods (<15%, dry matter basis) [
91
]. On average, Australian adults consume only
3–9 g
of RS
per day [
115
], which is significantly below the recommended daily intake of
15–20 g/day
of RS [
115
] considered essential for promoting bowel health benefits [
91
,
115
]. Currently,
there is no consensus on the effective dosage for different types of RS. In our study, we
incorporated RS4 at fractions ranging from 11% to 14% (by weight), which is similar to
previous rodent studies [
134
–
136
]. However, it is important to note that these doses, while
effective in rodents, are not directly comparable to the RS levels required in humans. Several
human studies have been conducted to explore the effective dosage of RS4 in increasing
SCFA production to exert beneficial effects. A 26-week clinical study by Upadhyaya
et al. [
131
] showed that dietary supplementation with 30% RS4 (v/vin flour) significantly
increased several faecal SCFA levels in individuals with metabolic syndrome, including
propionate, butyrate, valerate, and hexanoate. In another study, a dose–response trial was
conducted to investigate the effectiveness of different doses of RS4 (ranging from 10 to
50 g/day of RS4) in healthy participants [
132
]. Their findings indicated that the majority
of tested RS4 doses did not change the overall concentration of faecal SCFAs. However,
a significant increase in the concentration of propionate was observed at a minimum of a
very high RS4 dose of 35 g/day. SCFA concentrations in past studies were usually assessed
in faecal samples as a measure of colonic absorption and metabolism. However, it is
important to analyse plasma SCFA levels in order to assess their systemic effects. Therefore,
in our study, we analysed plasma propionate and butyrate concentrations and observed
no differences in any of our dietary groups. Of note, the concentrations of SCFAs in
plasma and faecal samples are known to be different [
137
–
139
]. It is important to consider
that SCFA concentrations can be influenced by diet, the gut microbiome, the and host
metabolism [
140
], which can lead to considerable variations in SCFA profiles in various
conditions and methods of analysis. Thus, further research is required to gain a better
understanding of the role that SCFAs play in yoyo dieting and RS supplementation, as well
as their potential use as biomarkers or therapeutic targets.
4.3. Beneficial Effects of Resistant Starch on Gut Microbiome
We observed that RS supplementation resulted in enriched Bifidobacterium and Parabac-
teroides, along with reduced bacterial diversity and distinct microbiota composition. The
increase in the relative abundance of Bifidobacterium aligns with findings from various stud-
ies [
79
,
141
–
143
], showing the effect of RS in significantly altering the gut microbiome. This
is likely to be advantageous as this genus is representative of microbial probiotics [
144
,
145
],
which are associated with stomach acid tolerance and carbohydrate metabolism [
146
–
148
],
anti-inflammatory properties [
149
], and the regulation of bowel movement [
131
,
150
–
152
].
Likewise, the enrichment of Parabacteroides is likely beneficial, as it has been linked to
improvements in conditions such as heparinase-exacerbated acute pancreatitis [
153
], neu-
roprotection [
154
], and the attenuation of inflammation [
155
]. Notably, Bifidobacterium and
Parabacteroides are both SCFA producers [
153
,
156
–
158
] that exert beneficial effects on human
health [
159
,
160
]. Moreover, we observed reduced bacterial diversity following resistant
starch supplementation in male mice. This was an unexpected finding as reduced diversity
is often associated with dysbiosis, particularly in obesity [
161
–
163
]. However, the notion
of diversity as an indicator of health status is being challenged, with a suggested focus
towards using health-associated taxa as a better metric for health status. There is also
emerging evidence showing that reduced diversity after dietary fibre intervention has been
associated with improved clinical outcomes [
164
–
166
]. Moreover, it is important to consider
that dietary fibre structure plays a key role in influencing the ability of gut bacteria to utilise
these substrates [
167
,
168
]. For example, low/intermediate-specificity dietary fibres, such
as RS2 [
168
], can be utilised and shared by many bacteria [
167
,
168
], which may promote
the growth of multiple microbes, resulting in increased bacterial diversity [
169
]. On the
Nutrients 2024,16, 3138 21 of 30
other hand, RS4, a type of dietary fibre with greater specificity than RS2 [
132
,
168
], can only
be utilised by a small group of bacteria [
167
,
168
], which will promote the increased growth
of fewer microbes, resulting in reduced diversity [
169
]. This is an important consideration
as reduced diversity is not always detrimental and can still be associated with improved
health status.
Furthermore, we found that supplementation with RS in male yoyo mice shifted
their gut microbiota profile closer to the control state following weight loss. However, the
microbiota profiles of yoyo RS and control mice were still significantly different by the end
of the dietary intervention, which is consistent with the findings by Thaiss et al. [
47
]. This
suggests a promising effect of RS supplementation in shifting the gut microbiota composi-
tion in the short–term but not in the long–term restoration of the gut microbiome. Of note,
microbiota restoration requires not only consuming a healthy (control) diet and achieving
successful weight loss but also long–term weight management to ensure sustainable weight
loss [
47
]. Moreover, we also observed significantly different genera associated with shifting
the gut microbiome composition of yoyo RS after weight loss. These include enriched
Mediterraneibacter and reduced Bifidobacterium,Parabacteroides, and Desulfovibrio, which may
have contradictory implications for human health. On one hand, enriched Mediterraneibac-
ter is likely not to be advantageous as this has been positively associated with increased
risks of developing obesity [
170
], liver cancer [
171
], and gut dysbiosis [
172
]. Likewise, a
reduction in Bifidobacterium and Parabacteroides might not be beneficial as they have been
associated with increased risks of irritable bowel syndrome [
173
–
177
] and obesity [
178
–
180
].
However, reduced Desulfovibrio is likely to be beneficial as these have been associated with
reduced risks of obesity [
161
,
181
,
182
], inflammatory bowel disease [
183
,
184
], fatty liver
disease [182], and diabetes [185].
In this study, while the effect of yoyo dieting and resistant starch on the gut microbiome
was investigated, one explicit limitation was the potential confounding effect of cohousing
animals (four mice per cage and two cages per diet treatment). However, single housing was
not feasible due to ethical concerns around compromising the biological and behavioural
well-being of animals, which could result in heightened emotional stress and a lack of
thermoregulation [
186
–
190
]. Furthermore, exploring gut microbiota changes using full-
length 16S long-read sequencing or shotgun sequencing will provide valuable insights as
these techniques provide higher taxonomy resolution and functional profiling.
5. Conclusions
In summary, our study reveals significant sex-dependent differences in metabolic
outcomes and microbiome signatures. While yoyo dieting leads to metabolic health im-
provement through short–term weight loss, it also results in a greater risk of obesity relapse.
Interestingly, increased resistant starch intake reduces body weight and may reduce the risk
of rapid weight regain, potentially via gut microbiome restoration in male but not in female
mice. However, the functional implications of resistant starch-induced microbiota changes
for metabolic health, weight regain susceptibility, and sex differences remain to be further
explored. This study emphasises the importance of improving the gut microbiome as an
attractive target for sustainable weight management and the development of interventions
for obesity and related conditions. Additional research is required to determine the effective
dosage of resistant starch in humans to support the development of evidence-based lifestyle
management initiatives to improve gut and metabolic health. Moreover, further long–term
human studies are warranted to elucidate the underlying mechanisms of how resistant
starch and yoyo dieting affect the gut microbiota and contribute to varying weight loss and
weight regain susceptibility outcomes between sexes.
Supplementary Materials: The following supporting information can be downloaded at: https://www.
mdpi.com/article/10.3390/nu16183138/s1. Refs. [
170
,
191
,
192
] are cited in the supplementary materials.
Nutrients 2024,16, 3138 22 of 30
Author Contributions: Conceptualization, L.R.R., S.L.M. and K.P.-N.; methodology, L.R.R., K.P.-N.,
S.L.M. and G.M.K.; animal experiments, K.P.-N. and L.R.R.; SCFA experiment, G.M.K., K.P.-N. and
L.R.R.; group allocation, K.P.-N. and L.R.R.; OGTT and ITT experiments, K.P.-N., L.R.R., S.L.M., T.C.
and K.A.-M.; histological experiment and examination, K.P.-N.; histological analysis, K.P.-N. and
M.Q.M.; formal analysis, K.P.-N. and M.O.; writing—original draft preparation, K.P.-N.; writing—
review and editing, K.P.-N., L.R.R., M.O., K.A.-M., S.L.M., G.M.K., M.Q.M. and T.C.; visualization,
K.P.-N.; supervision, L.R.R.; project administration, K.P.-N. All authors have read and agreed to the
published version of the manuscript.
Funding: This research received no external funding.
Institutional Review Board Statement: The animal study was conducted in accordance with the
guidelines of the National Health and Medical Research Council and approved by the Deakin
University Animal Ethics Committee (G01-2021; date of approval: 7 May 2021).
Informed Consent Statement: Not applicable.
Data Availability Statement: The authors confirm that the data supporting the findings of this study
are available within the article or upon reasonable request.
Conflicts of Interest: M.O’H. has a financial interest in Prevatex Pty Ltd., a company developing
probiotic-based biotherapeutics. Other authors declare no conflicts of interest.
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