FSN 281 Final Paper

Starvation and the Gut Microbiome 1
The Effect of Anorexia Nervosa and Non-Anorexic Starvation on the Gut Microbiome
Sarah Tu, Jennifer Zielke, Caitlyn Lew
California Polytechnic State University, San Luis Obispo, CA
FSN 281 Section 1
June 29, 2021

Introduction
The gut is home to a variety of microbiota that maintain a symbiotic relationship with the
host, carrying out processes such as energy metabolism, immune system maintenance, and
communication with the central nervous system. Gut dysbiosis is the disruption of this
microbiome. Because dysbiosis has such a broad definition, there is limited research detailing
dysbiosis' prevalence in the United States. Common diseases in the US that are associated with
gut dysbiosis include digestion-related diseases such as irritable bowel syndrome (IBS) and
inflammatory bowel disease (IBD), obesity, cancer, and Type 2 diabetes mellitus (T2DM). It has
also been observed in neurodegenerative diseases such as Parkinson’s disease (PD), Alzheimer’s
disease (AD), multiple sclerosis (MS), and depression [1-4]. Causes of gut dysbiosis include
changes in microbial gut populations, environmental factors, and pathogen exposure. Changes in
microbial populations can occur as a result of the host changing lifestyle habits, such as diet,
exercise, and stress management. Gut dysbiosis can be diagnosed through biopsy of fecal
samples, endoscopy, luminal brushing, and laser capture microdissection [5]. As the literature
has established, gut dysbiosis has a profound effect on overall health in the course of several
medical conditions.
Anorexia nervosa (AN) is an eating disorder with a progression characterized by
voluntary self-starvation, distorted body image, and low body weight. In the Diagnostic and
Statistical Manual of Mental Disorders (DSM-5), the diagnostic criteria for AN includes
restriction of energy intake leading to low body weight, intense fear of weight gain, and body
image distortion [6]. The etiology of AN is not well understood. AN is thought to be primarily
mental (i.e. negative body image or concerns with body weight), but evidence of distrubed
appetite and feeding pathways suggest that AN may be biologically driven. Approximately 60%
of the variance in AN is due to genetic or epigenetic origins, and psychosocial factors are also
significant in AN development [7]. Due to genetic associations between AN and low body mass
index (BMI), low incidence of type 2 diabetes (T2DM), low fasting insulin, and high
high-density lipoprotein (HDL) cholesterol, AN is considered to have metabo-psychiatric origins
[8].
AN leads to severe endocrine and somatic consequences including increased cortisol
levels, amenorrhea, low leptin and triiodothyronine levels, and increased ghrelin levels,
negatively impacting energy homeostasis and appetite regulation in AN patients. [9, 10]. Stice
and Bohon found that between 0.9% and 2.0% of females and 0.1% to 0.3% of males in the
United States (U.S.) will develop AN [11]. Hudson et al. found that 0.9% of women and 0.3% of
men in the U.S. develop AN during their lives [12]. An ongoing study in Minnesota has found
AN incidence to have increased over the last approximately 50 years in females ages 15 to 24
[13]. In adolescent females, AN has an incidence of 100-200/100,000 persons per year, which is
similar to the incidence of type 1 diabetes (T1DM) [7, 14]. AN is one of the most prevalent
chronic illnesses for adolescent girls [7]. In the UK and Germany, AN hospital admissions rose
substantially between 1990 and 2011, becoming a greater burden on secondary health care,
particularly due to multiple admissions per AN patient [15]. For AN patients, relapse rates are
high due to AN’s chronic course, and its mortality rate is the highest of all psychiatric disorders
[16]. The emotional consequences for AN patients and their families are approximately
equivalent to those of schizophrenia and depression patients [17].
In several studies, researchers have found that the gut microbiota is altered in patients
with AN. Such alteration is considered gut dysbiosis [18-25]. The overall effect of chronic
energy restriction in AN individuals is a reduction in the diversity of the gut microbiota, which is
associated with poor clinical outcomes [26]. Chronic caloric deprivation outside of AN (hereafter
starvation) also results in a reduction of diversity of gut microbiota. Further, animal data suggests
that the lack of diversity in the gut microbiome actively contributes to malnourishment in the
host. One study found that a gut microbial transplant from human subjects with Kwashiorkor
(severe protein-energy malnutrition) induced altered protein and carbohydrate metabolism in
mice [27, 28].
Research has also shown that patients with AN have microbiomes constant with those of
patients with suppressed appetite, depression, and anxiety. Studies of fecal transplants in animal
models have produced results that support the hypothesis that the gut microbiome can affect
psychological health. These studies involved collecting fecal transplants from patients with
depression and inducting them into animal models, then observing if the models exhibited
similar symptoms [2, 29]. Because AN is foremost a psychological disorder, it is important to
understand how gut health can affect the host psychologically in order to improve treatment
methods. The effects of caloric deprivation in AN and starvation on the gut microbiome are
underexplored in the literature. Further, any potential differences between the effects of caloric
deprivation in AN and starvation on the gut microbiome, and thus the significant impacts the gut
microbiota have on health outcomes, are not well established in the literature.
It’s important to examine the effect of starvation and negative energy balance on the gut
microbiota, as starvation, whether intentional or not, may also impact the energy metabolism of
gut bacteria. Inadequate food intake directly affects energy consumption and production of
bacteria [30]. Furthermore, AN and starvation can also affect the psychological state of the host.
Because a healthy gut microbiome has an immense effect on a host’s metabolic and energy
function, understanding the importance of its role and how AN and starvation alter it is essential
as AN becomes more prevalent. This review will investigate the effects of starvation in AN and
the effects of general starvation on the gut microbiome, compare the two, and investigate how
the differences between them may impact treatment of malnutrition.
Methods
This review includes a combination of review articles and studies for background
information and an analysis of six original research articles including cross-sectional
comparative studies and controlled intervention studies of humans and animals, specifically rat
and mouse species. The database PubMed was used to identify all original research articles.
Excluded from the search were case-studies, and studies done on non-vertebrates. The articles
selected for analysis were all published in the last ten years, with the majority of them being
published in the last five years. Dates of publication range from December 2015 to December
2020. Studies that were excluded included non-mammalian studies, individual case studies, and
studies that didn’t focus directly on alteration of the gut microbiome. The connection between
eating disorders, specifically AN, and the health of the gut microbiome has become a more
researched topic in recent years, which allowed the inclusion of fairly recent studies and
information in this review. Key terms used in the PubMed search included “anorexia nervosa,”
“gut dysbiosis,” “calorie restriction,” and “gut microbiota.” Searches were conducted between
June 24 and July 1, 2021.
Results
Six primary studies examining the effect of AN and starvation on gut microbiome health
were selected. Four were interventional trials, two on the effects of calorie deprivation on obese
human subjects’ gut microbiota and two on the effect of anorexia models on rodent gut
microbiota. The remaining two were cross-sectional comparative studies, in which researchers
compared the gut microbiomes of human AN patients and healthy controls. Overall, all six of the
studies found that caloric restriction has a significant, often detrimental, effect on the health and
diversity of the gut microbiota. In this review, the studies were divided into three categories
based on their subjects and designs: interventional trials on rodents, interventional trials on obese
human subjects without AN, and cross-sectional comparative studies contrasting AN patients and
controls. See the Results Summary Table at the end of the section for main findings.
Interventional Trials on Obese Subjects
A study conducted by Heinsen et al. examined the effect of a very low-calorie diet
(VLCD) on the gut microbiome and metabolism of obese individuals over a three month period,
followed by a period of weight maintenance [36]. The study was a randomized interventional
trial on human subjects. Subjects were between 20 and 75 years of age, had a BMI between 27
and 40, and were non-smokers or smokers with stable smoking habits of at least 6 months. Two
control groups were used, one lean control group (LC, BMI < 25 kg/m2) and one obese control
group (OC, BMI > 30 kg/m2). Fasting insulin and glucose levels were taken prior to the start of
the intervention. During the first half of the intervention, the weight loss phase, the VLCD group
followed a formula-based meal plan of roughly 800 calories. The macronutrient distribution of
total calories consisted of 50% carbohydrates, 33% protein, and 17% fats. During the second half
of the intervention, the weight maintenance phase, the VLCD group was slowly weaned off the
formula-based diet in a 5-week period before fully transitioning to a conventional food diet of
1600 calories per day. Fecal samples were collected and bacterial DNA was extracted using the
QIAamp DNA stool kit. The Bacterial 16S rRNA genes were sequenced and ran through the
Greengenes OTU database for identification. These processes were conducted before the
intervention (0 months), at the 3-month checkpoint (VLCD intervention), and at the 6-month
checkpoint (weight maintenance). Researchers performed a PERMANOVA analysis and used the
Bray-Curtis dissimilarity to measure variability within the bacterial groups during the
intervention. The change in variance between the baseline and the 3-month checkpoint was
significant, with 6.22% of the gut microbiota variance being explained by the VLCD diet (p <
0.048). The 3-month to 6-month period had fewer changes in variance and was insignificant.
Figure 1: Bray-Curtis dissimilarity capscale showing the explained variance at 0-months

(baseline), 3-months (dietary intervention), and 6-months (weight maintenance period). Data
from [36].
Activity of metabolic pathways was measured as well. Results showed that the riboflavin
pathway activity decreased significantly during the VLCD period, then increased slightly during
the weight maintenance period (p < 0.0078 and p < 0.039, respectively).
Figure 2: Riboflavin metabolism activity during the three checkpoints. Data from [36].
A study conducted by Dong et al. explored the differences of a normal protein diet (NPD)
and a high protein diet (HPD) on the gut microbiome [37]. The Dong et al. study was a
randomized interventional human study. Subjects were between 20 and 75 years of age, had a
BMI between 27 and 40, and were non-smokers or smokers with stable smoking habits of at least
6 months. Subjects were randomly assigned to groups, with the NPD group serving as the control
group. The NPD had a macronutrient distribution of 15% protein, 55% carbohydrate, and 30%
fat, whereas the HPD consisted of 30% protein, 40% carbohydrate, and 30% fat. The diet
intervention had two phases, with the first phase being two weeks of the assigned diet with no
caloric restriction, then six weeks of the assigned diet with a 500 calorie deficit. Patients were
provided home sampling fecal kits and samples were collected during weeks 1, 2, 4, 6 and 8.
Bacterial DNA was extracted and sequenced, then imported into the SILVA 132 database for
identification. For statistical analysis, alpha diversity was calculated using the Shannon index,
whereas the beta diversity was calculated using QIIME 2. Baseline samples showed that there
was little to no difference between alpha and beta diversity. Alpha diversity decreased initially in
both groups, however only the HPD group had increased diversity at the end of the 8 weeks. The
most impacted genera were Akkermansia spp., Bifidobacterium spp., and Prevotella_9. While
Akkermansia spp. and Bifidobacterium spp. were elevated from baseline, Prevotella_9 was
decreased from baseline.
Trials on Animal Subjects
In a cross-sectional, randomized study by Breton et al., researchers investigated whether
models of activity based anorexia (ABA) altered the proteome of gut microbiota relevant to ATP
and acetate production in mice [30]. The 17-day study was conducted using 32 male C57 B1/6
mice, who were divided into four groups based on body weight. The groups were ABA,
feeding-time restriction (FTR), ad libitum-fed controls with physical activity (CTPA), and
without (CT). The independent variables were food restriction and physical activity. ABA and
CTPA were placed in individual cages with an activity wheel, and their activity was continuously
recorded throughout the study. FTR and control groups were placed in individual cages without
an activity wheel. All mice had free access to water and a standard diet on days 1-5, then food
access became progressively limited in ABA and FTR groups from day 6 to day 9, until the end
of the experiment. Food consumption was measured when food was removed from the cages.
Body weight, water intake, and food consumption were measured daily. On day 17, mice were
sacrificed and colon pellets were immediately frozen at -80˚C for proteomic analysis of gut
microbiota. In vitro adenosine triphosphate ATP production was measured using an ATP
fluorometric assay kit. The bacterial proteins from feces of ABA, CT, FTR, and CTPA mice were
put in duplicates into the wells and adjusted with ATP assay buffer, which was followed by an
incubation period. Further analysis of the capacity of bacterial proteins from feces to produce
ATP in starved mice was conducted by incubation in vitro for two hours. No significant
difference was found among the groups. The adjustment of ATP production by body weight of
mice did not result in significant differences among the four groups. To compare proteome
profiles of the four groups, 2-D electrophoresis gels were run with the total protein samples
extracted from feces in the colons of CT, FTR, and ABA groups. Between CT and FTR groups,
16 proteins were overexpressed in the CT group, and 4 in the FTR group (p < 0.05). The 2-D
gels further revealed that 26 proteins were different between the FTR and ABA groups ( p <
0.05). Increased levels of phosphoglycerate kinase, the ATP-producing glycolytic enzyme
belonging to Clostridium, were detected in the ABA group. Other proteins from Clostridium and
Lachnospiraceae were increased in the starvation groups.
A study by Trinh et al. was conducted to analyze whether different starvation and activity
conditions altered fecal microbiota in rats [31]. This was a randomized controlled animal study;
the researchers were not blinded to the group assignments, but all analyses were conducted blind.
To emulate the higher prevalence of AN in adolescent women, the subjects were 49 4-week old
female Wistar rats who were divided into four groups. The four groups were the control animals
(C), control animals with running wheel (CRW), animals with reduced bodyweight (RBW), and
animals with reduced bodyweight and running wheel, which mimicked ABA. The independent
variables were food reduction and food reduction combined with the running wheel. The
dependent variables included alpha diversity and beta diversity parameters and bacterial genera
abundance. All rats were housed under specific pathogen-free conditions and in a maintained
environment. There was a 10 day acclimatization period for all animals to habituate to the
assigned conditions and have normal access to food. After acclimatization, food availability was
reduced to 30% of the daily food intake for RBW and ABA until they lost 25% of their body
weight. Food intake was maintained to keep the body weight stable at -25%. All control groups
had ad libitum access to food throughout the experiment. After day 30 of chronic starvation,
fresh fecal samples were collected and stored in sterile microtubes at -80˚C. The genes were
amplified using polymerase chain reaction (PCR) to be purified via gel electrophoresis and gel
extraction. This study found a significant impact of food reduction on specifically alpha diversity
after chronic starvation (p < 0.022). In food restricted animals, alpha diversity was increased
compared to control groups. RBW and ABA groups had significantly different beta diversity
from those in the C and CRW groups (p < 0.001). The results further indicated that of the 45
bacterial genera studies, food restriction had a statistically significant effect on six of the genera.
Figure 3: Relative abundances of specific genera after starvation. Data from [30].
The six bacterial genera were Prevotella (p < 0.0001), Odoribacter (p < 0.013),
Lactobacillus (p < 0.017), Akkermansia (p < 0.0001), Bifidobacterium (p < 0.032), and
Ruminococcus (p < 0.032). After starvation, relative abundance of Prevotella (p < 0.0004)
decreased while Odoribacter (p < 0.0003), Lactobacillus (p < 0.02), Akkermansia (p < 0.013),
Bifidobacteirum (p < 0.048), and Ruminococcus (p < 0.048) increased in animals with reduced
body weight. Running wheel activity (RWA) resulted in a significant presence of Akkermansia (p
< 0.022) and Bifidobacterium (p < 0.047). For Bifidobacterium, food restriction combined with
RWA had a significant effect, revealing the influence of exercise on the gut microbiota.
Cross-Sectional Studies Comparing AN Patients and Control
In a cross-sectional comparative study, Morita et al. compared the fecal microbiota of
female AN patients with both restrictive (ANR) and binge-purge (ANBP) AN subtypes to those
of age-matched healthy controls to determine whether AN patients had gut dysbiosis [24]. The
participants’ gut microbiota were compared using Yakult Intestinal Flora-SCAN (YIF-SCAN®)
based on rRNA-target RT-quantitative PCR technology. The participants were Japanese AN
patients admitted to Kyushu University Hospital between 2010 and 2013; specifically 25 female
patients (14 ANR, 11 ANBP), as well as 21 age-matched, healthy female volunteers as controls,
excluding volunteers with a history of digestive diseases (e.g. IBD, IBS). AN patients underwent
structured interviews and their AN subtypes were diagnosed according to the DSM-5. On a
single morning, blood samples were collected from all participants to determine serum levels of
albumin, blood urea, nitrogen, electrolytes, creatinine, aspartate aminotransferase, alanine
aminotransferase, total cholesterol, triglycerides, and glucose. Latex nephelometry was used to
determine high sensitivity C-reactive protein levels. For each bacterium, the detection rate was
evaluated by calculating the ratio of the participants in each group whose feces contained the
bacterium to the total number of participants whose feces did not. Fecal samples were collected
from all participants and RNA fractions were extracted from the feces through processes
described previously [32-34]. YIF-SCAN® was used to examine the gut bacterial group
composition based on 16S or 23S rRNA-targeted RT-qPCR technology. The presence of fecal
organic acids was determined through methods previously described [35]. The fecal samples
were homogenized in 0.15 mol/L perchloric acid and the resulting suspension was centrifuged
for 10 minutes. A high-performance liquid chromatography system was used to measure the
concentrations of organic acids in the sample (including acetic and propionic acids), and fecal pH
was checked using the IQ 150 pH/thermometer. The nonparametric Kruskal-Wallis test was used
to examine differences in serum chemical parameters and bacterial counts between the ANR,
ANBP, and control groups. Comparisons between the AN group and the control group were done
using Fisher’s exact test and subjected to the Bonferroni correction. The log-transformed
bacterial count was used for principal component analysis (PCA). The programming language R
3.1.1 was used to apply PCA to determine the data sets which included data on the Clostridium
coccoides group, Clostridium leptum subgroup, Bacteroides fragilis group, Bifidobacterium,
Atopobium cluster, Prevotella, Enterobacteriaceae, Enterococcus, Staphylococcus,
Streptococcus, Clostridium difficile, Clostridium perfringens, and total Lactobacillus.

Figure 4: Principal component analysis (PCA) of bacterial counts in healthy female controls, 14
restrictive anorexia nervosa (ANR) patients, and 10 binge-eating anorexia nervosa (ANBP)
patients. Black, red, and purple plots show data for the healthy female controls, ANR patients,
and ANBP patients, respectively. The colored ellipse represents 50% of the samples. Arrows
indicate the characteristic vectors of the upper 4 factor loadings. The numbers in parentheses
represent the proportion of variance. Data from [24].

Weight (31.4 ± 3.6 kg, p < 0.001), BMI ( 12.8 ± 1.3 kg/m2, p < 0.0001), and serum
C-reactive protein levels (0.04 ± 0.06 mg/dL, p < 0.0003) were significantly lower in the AN
patients (and for both ANR and ANBP subtypes) than in the controls. The AN patients had
significantly higher serum aspartate aminotransferase (38.9 ± 22.9 IU/L, p < 0.0001) and alanine
aminotransferase (40.6 ± 34.0 IU/L, p < 0.0001) levels than the control group. Both the ANR and
ANBP subgroups had significantly lower potassium (4.2 ± 0.4 mEq/L, 3.2 ± 0.6 mEq/L, p <
0.0001) in the fecal samples. The counts of total bacteria (10.5 ± 0.5, p < 0.0002), as well as
Clostridium coccoides group (9.3 ± 0.6, p < 0.0001), Clostridium leptum subgroup (9.6 ± 0.6, p
< 0.0006), Bacteroides fragilis (9.6 ± 0.6, p < 0.0001) group, and Streptococcus (8.2 ± 0.8, p <
0.0003) were significantly lower in the AN patients than in the control groups. The ANR and
ANBP subgroups had lower counts of Bacteroides fragilis (9.6 ± 0.7, 9.5 ± 0.6, p < 0.0007) and
the ANR group had significantly lower counts of the Clostridium coccoides group (9.3 ± 0.6, p <
0.0003) than the control. The AN group had significantly lower detection of the Lactobacillus
plantarum subgroup than the control group. Clostridium difficile was present in 45% (5 of 11) of
the ANPB patients’ feces ( p < 0.0002). In contrast, no patients in the ANR subgroup or the
control group had detectable levels of C. difficile. However, the differences between the ANBP
and AN groups were not statistically significant. The AN group had significantly lower
concentrations of acetic (30.7 ± 13.2, p < 0.0003) and propionic (9.3 ± 4.8, p < 0.001) acid in
their fecal samples than the control group did.
In their cross-sectional comparative study, Mack et al. comprehensively investigated the
fecal microbiota and short-chain fatty acids (SCFA) in AN patients before and after weight gain
and compared them to those of normal-weight (NW) participants to examine the extent to which
AN patients’ gut microbiota were perturbed compared to NW controls and whether those
perturbations recovered after weight gain and/or normalization of eating behavior [22]. Data
collected at the beginning of the study was part of the T1 or ANT1 group and data collected after
the intervention was part of the T2 or ANT2 group. The participants were 55 female AN patients
at baseline (T1) and 44 patients after intervention (T2) between September 2011 and October
2012. These subjects had a mean age of 23.8 ± 6.8 years, a mean BMI of 15.3 ± 1.4 kg/m2 at T1,
and a mean BMI of 17.7 ± 1.4 kg/m2 at T2. Of the AN patients, 39 had ANR and 16 had ANBP.
As controls, 55 healthy female NW participants were selected. The NW participants were
matched for age and gender to the AN patients at T1. The NW controls had a mean age of
23.7 ± 6.7 years and a mean BMI 21.6 ± 2.0 kg/m2. AN patients were recruited from an AN
inpatient program at Schön Klinik Roseneck, Prien, Germany that aimed to increase BMI and
normalize eating habits for patients with a pre-existing DSM-5 AN diagnosis. Female
participants were 14-39 years old with BMI less than 18 kg/m2 in adults or less than 10% of the
expected weight in adolescents.The exclusion criteria for NW controls was BMI less than 18
kg/m2 in adults or less than 10% of the expected weight of adolescents. Stool samples of AN
patients were collected early in the inpatients’ stay. The samples were frozen at - 80 °C. NW
controls collected fecal samples at home using the same process. A blind analysis of the SCFA
was conducted. Upon inclusion in the study, AN participants were guided through a 24-hour food
intake recall and a food frequency questionnaire (FFQ) by trained interviewers before fecal
sampling. During the programs, AN patients received one of four diet plans, a 2000 kcal/d diet, a
2400 kcal/d diet, a 2800 kcal/day diet, or a 3050 kcal/d diet that was 45% fat, 40% carbohydrate,
and 15% protein. The multiple source method (MSM) was used to estimate typical total food,
calorie, fat, carbohydrate, and protein intake for NW controls. A validated gastro-questionnaire
was used to assess the frequency and severity of 27 gastrointestinal (GI) symptoms. At T1, the
AN patients and NW controls assessed their GI symptoms including upper GI-symptoms (UGIS)
(sensation of lump in the throat, regurgitation of food, difficult swallowing, nausea, vomiting,
heartburn, and bloating) and lower GI-symptoms (LGIS) (abdominal pain, bowel noise, frequent
stools, rare stools, fecal soiling, loose or watery stools, straining during bowel movement, and
feeling of incomplete evacuation). These symptoms were rated on a scale of 1 (no distress) to 5
(severe distress). Severity scores were calculated. At T2, the AN patients completed the
questionnaire again.
The LotuS pipeline was used to filter, denoise, remove chimeric sequences and cluster
sequences in Operational Taxonomic Units (OTUs) at 97% similarity. To evaluate differences in
the relative abundance of genera between AN patients and NW controls, Spearman rank’s
correlation were calculated. The measures of microbial species diversity within communities
(alpha-diversity), included observed OTUs (observed richness), and Shannon diversity index.
Diversity across samples (beta diversity) was determined by unweighted and weighted UniFrac
distance. The number of OTUs present in a fraction of subjects per group ranging from 75% to
100% were calculated to examine differences in the microbial core of AN patients at T1 and T2
and the NW controls. Subsequent assessment was performed to determine which OTUs were
shared in the cores of AN patients before and after weight gain and NW participants.
Distance-based redundancy analysis (db-RDA), an extension of PCoA, was performed with the
capscale function. Variance partitioning with db-RDA of Bray-Curtis distances of all samples of
AN patients at baseline was investigated to determine the extent to which anorexia subtype
(ANR or ANBP) and total energy, fiber, protein, and fat intakes determine microbial community
structure. The extent to which disease status explained the microbial community structure was
determined using db-RDA analysis of BC distances of samples from AN patients at T1 and NW
participants.
SCFAs in the fecal samples were analysed using gas chromatography. For determination
of the SCFAs, an external standard was used. For statistical analysis of all parameters, the
differences in data between AN patients at T1 and T2, and as compared to the NW controls were
determined using the Wilcoxon signed-rank test and Mann-Whitney-U-test.
BMI differed by 6.3 kg/m2 between AN patients before weight gain and NW controls,
and by 3.9 kg/m2 between AN patients after weight gain and NW controls. At T1, the energy and
macronutrient intake of AN patients and NW participants was compared and, although most AN
patients avoided the intake of many foods, their fruit, vegetable, and whole grains consumption
was similar to that of NW controls, resulting in similar daily fiber intake to that of the NW
controls. During their inpatient stays, AN patients were required to follow their prescribed diet
plans, resulting in high energy, fat, and fiber intake when compared to NW participants.
Compared to NW controls, AN patients had higher levels of LGIS and particularly
UGIS. While symptom scores of UGIS and LGIS improved at T2 overall, for the majority of
individual UGIS, the severity of symptoms including food regurgitation, heartburn, and
abdominal bloating did not lessen from T1 and T2. However, many LGIS improved between T1
and T2. Abdominal pain, bowel movements and incomplete evacuation were more severe in AN
patients at T2 than NW controls. In both the AN patients and NW controls, the predominant
phyla were Bacteroidetes, Firmicutes, Actinobacteria, Proteobacteria, and Verrucomicrobia. The
relative abundance of Bacteroidetes was significantly lower in AN patients at T1 than in NW
controls and it further decreased in AN patients after weight gain at T2. The abundance of
Firmicutes significantly increased in AN patients between T1 and T2. At T2, the abundance of
Firmicutes was higher than that of the NW controls.

Figure 5: Taxonomy at phylum and genus level for anorexia nervosa patients (AN) before (T1)
and after (T2) weight gain compared to normal-weight participants (NW). Data from [22].
In AN patients at both T1 and T2, Actinobacteria levels were high compared to NW
participants. Verrucomicrobia levels were higher in AN patients at T1, but decreased after weight
gain. The majority of the significant differences in the relative abundance of bacterial genera
between NW participants and AN patients were in the Firmicutes phylum. Archaea, primarily of
the genus Methanobrevibacter, were detected in 22% of ANT1 patients, in 14% of ANT2
patients, and in 15% of NW participants. The relative abundance of archaeon was statistically
significantly higher in ANT1 patients (0.10 ± 0.05, p < 0.004) compared to NW participants
(0.01 ± 0.03, p < 0.004). The core microbiome was defined as a minimum of 90% of samples
containing specific OTUs. The core microbiome contained 41 OTUs that were shared by the
ANT1, ANT2, and NW groups. NW participants shared 54 core OTUs with ANT2 patients, 13
of which were not present in ANT1. These OTUs included those in the genera Coprococcus,
Dorea, and Clostridium cluster XIVa. Only the core of ANT1 patients had Clostridium cluster XI.
There were four OTUs which were shared between the ANT1 and ANT2 patients (two of which
were of the genus Bifidobacterium), while bifidobacteria were not present in the core of NW
participants. When looking at the core microbiome over a window of different maximum
factions of samples, ANT1 patients had the lowest core size and ANT2 patients had the highest.
The number of OTUs increased significantly between T1 and T2 for AN patients. No differences
in OTUs to NW controls were observed. Biodiversity, measured with the Shannon index, was
significantly increased in ANT2 patients compared to that of NW controls.
The beta diversity of fecal samples was assessed using the unweighted UniFrac and
Bray-Curtis distances. In AN patients, the intra-individual dissimilarity in microbial composition
(the difference between the T1 and T2 samples for a single patient) was significantly lower than
the dissimilarity between ANT1, ANT2, and NW groups overall. Thus, the fecal microbiota
composition of AN patients after weight gain is more similar to the microbiota composition of
the patients’ own fecal samples at admission than it is to the microbiota composition of other
individuals. Total SCFA levels were normal in AN patients while the concentrations and
proportions of branched chain fatty acids (BCFA) increased before and after weight gain in AN
patients compared to NW controls. Overall, the researchers found that there were significant
differences between the combined intestinal microbiota of AN patients before and after weight
gain and NW controls, indicating the presence of gut dysbiosis in AN patients. However, weight
gain in AN does not improve gut microbiota diversity, BCFA profiles, or GI complaints.
Results Summary Table
Study # Age Independ Dependent Study Design Results

Participan (years) ent Variable
ts Variable
Heinsen 18 Mean: VLCD Diversity in Randomized Increase in

et al., VLCD for 3 gut interventional microbiota
2016 = 47.0 months microbiota study variance during
(range: followed Metabolism the VLCD
38.8-54 by 3 efficiency intervention
.5) months of
LC = weight Insignificant
46.0 maintena changes during
(range nce the weight
37.5-50 maintenance
.5) period
OC =
50.0 Decreased
(range: riboflavin
38.5-54 metabolism
.0) during 3 and 6
month
checkpoints (p
= 0.0078, p =
0.039,
respectively)
T. Dong 80 NPD: High Diversity of Randomized Initial decrease

et al., 55.7± protein the gut interventional in alpha
2020 0.4 diet for 8 microbiome human study diversity in
HPD: weeks both groups,
55.9± followed by
10.1 increase in
alpha diversity
in HPD group
Akkermansia
spp.,
Bifidobacteriu
m spp., and
Prevotella_9
were most
abundant in
HPD group
Decrease in
Prevotella_9
by the end of
the 8 weeks
J. 32 N/A Food Pellets and Cross-sectional, Sixteen

Breton restriction feces from randomized overexpressed
et al., Physical the colons interventional proteins in the
2019 activity of the mice animal study CT group, 4 in
level to determine the FTR group
diversity of
gut Difference of
microbiota 26 proteins
between the
FTR and ABA
groups
S. Trinh 49 4 Food ɑ-diversity Randomized Food restricted

et al., weeks reduction and controlled animals,
2020 Food ϐ-diversity interventional ɑ-diversity was
reduction parameters animal study increased
combined and compared to
with bacterial control groups
running genera
wheel abundance
activity RBW and

ABA groups
had
significantly
different
ϐ-diversity
from those in
the C and
CRW groups
Of the 45
bacterial
genera studies,
food restriction
had a
statistically
significant
effect on 6 of
them
C. 46 (25 AN Control AN Diversity of Cross-sectional AN patients

Morita patients - : 31.5 ± (ANR the gut comparative had
et al., 14 ANR, 7.4 and microbiota Study significantly
2015 11 ANBP, years ANBP) in as compared lower counts of
and 21 the AN to the total bacteria,
healthy AN: patients C. coccoides
controls 30.0 ± group, C.
10.2 leptum
years subgroup, B.
fragilis, and
Streptococcus
than controls
Acetic acid and

propionic acids
were
significantly
lower in the
AN group than
the control
group
Bacteroides
fragilis groups
in both the
ANR and
ANBP groups
and the
Clostridium
coccoides
counts in the
ANR group
were
significantly
lower than that
of the controls
PCA results
showed that
the patterns of
gut microbiota
in the AN
group differed
from that of the
control group
I. Mack 110 (55 NW: AN Diversity of Cross-sectional Bacteroidetes

et al., AN 23.7 ± before the gut comparative abundance was
2016 patients at 6.8 weight microbiota, study somewhat
T1, of years gain at T1 PCFA lower in ANT1
which 44 and after profiles, and patients and
were also AN: weight gastrointesti majorly lower
AN 23.8 ± gain at T2 nal distress in ANT2
patients at 6.7 in AN symptoms patients
T2, and 55 years patients compared to
NW NW controls
controls)
The abundance
of Firmicutes
significantly
increased in
AN patients
between T1
and T2. At T2
the abundance
of Firmicutes
was higher
than that of the
NW controls
AN patients at
T1 and T2 had
higher
Actinobacteria
levels that
were high
compared to
NW
participants
Archaea of the
genus
Methanobrevib
acter, were
detected in
22% of ANT1
patients, in
14% of ANT2
patients and in
15% of NW
participants
The ANT1,
ANT2, and
NW groups
shared 41
OTUs
ANT1 and
ANT2 groups
shared 4 OTUs
in the
Bifidobacteriu
m genus. The
NW group
lacked
bifidobacteria
ANT2 and NW
groups had 13
OTUs not
present in the
ANT1 group in
the
Microbiota
diversity was
greater for the
ANT2 group
than the NW
group
The microbial
composition
intra-individual
dissimilarity
between ANT1
and ANT2 was
lower than the
similarity
between the
ANT1, ANT2,
and NW
groups overall
Discussion
This review investigated the effect of AN and other starvation methods on the gut
microbiome and gut dysbiosis. Six primary studies were found through the PubMed database
using key terms such as “anorexia nervosa,” “gut dysbiosis,” “calorie restriction,” and “gut
microbiota.” The studies were divided into three categories: interventional studies on obese
subjects, randomized interventional trials on animal models, and cross-sectional studies
comparing AN patients with a control group. Results were mixed on the effect of caloric
restriction on gut microbiota. Four of the studies found that once caloric restriction began, alpha
diversity increased, but one of the cross-sectional AN studies showed that there were significant
differences between the gut microbiota of the AN patients and the healthy control subjects,
suggesting that long term caloric restriction may result in gut dysbiosis.
The effects of caloric restriction on gut microbiota varied between studies. Microbiota
status was analyzed using two methods, alpha and beta diversity. Alpha diversity was measured
by analyzing the diversity within a sample, whereas beta diversity was measured through
comparison between two groups. The obesity intervention studies conducted by Heinsen et al.
and Dong et al. and the animal model study conducted by Trinh et al. all demonstrated an
increase in alpha diversity with caloric restriction [31, 36, 37]. The AN studies conducted by
Mack et al. and Morita et al., however, both indicated that there was no difference in alpha
diversity between AN patients and healthy controls [24] and that there were decreased counts of
several groups of microbiota [22].
Obesity as a possible cause of gut dysbiosis may explain why the obesity intervention
studies resulted in a positive result for the subjects [70]. The increase in alpha diversity was
likely a beneficial effect that occurred with weight loss. Furthermore, because the intervention
was short-term, the effects on the gut microbiota wouldn’t be as extreme compared to the gut
microbiome of an AN patient that had maintained their caloric-restrictive lifestyle for an
extended period of time. However, the data does show that an improvement in health leads to an
improvement in gut microbiota diversity. This is seen in both obesity studies [36, 37], in which
the subjects lost weight, as well as the study conducted by Mack et al. which involved a weight
gain intervention in AN patients. The weight gain led to an increase in OTUs and overall alpha
diversity in the patients [24].
The literature currently focuses primarily on the role of gut microbiota in obesity with
respect to weight gain [39]. There have been studies conducted regarding undernutrition in
children and the role of the gut microbiota [33, 34]. However, the literature is largely lacking in
studies investigating the gut microbiota of AN patients. In their study, Mack et al. found
profound differences in the microbial composition between AN patients before weight gain
intervention and NW controls, and differences between AN patients after weight gain and NW
controls. Essentially, weight restoration in AN patients did not return the gut microbiota
population to a state congruent with that of an unafflicted control. The researchers found that,
although the GI symptoms improved overall in AN patients after the intervention, the mean three
month period of weight gain was not sufficient to eliminate the GI symptoms. Thus, the gut
microbiota is impacted by the course of AN and weight restoration. Though such treatment does
improve microbiota diversity, it does not return diversity to a pre-AN state.
Studies that investigate the effects of caloric restriction on the gut microbiome can be
helpful in the diagnosis and treatment of AN, as some studies have suggested that the gut
dysbiosis that is caused by AN hinders recovery through symptoms such as decreased appetite
and lack of hunger cues [38]. Current research explores the gut-brain axis and how treating
dysbiosis can affect neurodegenerative diseases, and because AN is first and foremost a
psychological disease, it may be helpful in understanding how the gut dysbiosis experienced by
AN patients causes them to continue their behavior.
Typically, lower bacterial diversity is considered a detriment to intestinal health. It has
been linked to several other diseases [40]. The literature includes several examples of caloric
restriction leading to decreased microbiota diversity, including in neonatal mice subjected to
reduced lactation [41], malnourished Bangladeshi children [33, 52], and a small sample of AN
patients [2]. In their study, Mack et al. found that microbial richness (number of species in a
community) and evenness (relative abundance of species) was relatively similar in AN patients
before treatment and NW controls [22]. However, this was likely due to the normal level of fiber
intake and the normal distribution of energy derived from each macronutrient for AN patients.
The combination of these factors allowed the normal-appearing alpha-diversity in AN patients.

Bacteroides perform carbohydrate fermentation in the gut, which results in fatty acids
that are utilized by the host as a daily energy source. In the Mack et al. study, researchers
observed a reduced abundance of Bacteroidetes at T1 [22]. This is in concordance with
decreased Bacteroidetes found in neonatal mice under reduced lactation [41], and malnourished
Bangladeshi children [52]. Morita et al. found that AN patients had lower counts of Bacteroides
fragilis (a Bacteroidetes genera) [24].
Firmicutes are implicated in mucin degradation [43]. In the Mack et al. study,
Anaerotruncus species (of the Firmicutes phylum) were increased in AN patients prior to weight
gain. However, Firmicutes significantly increased in AN patients after weight gain [22].
Meanwhile, Morita et al. found that counts of Clostridium coccoides group, Clostridium leptum
subgroup, and Lactobacillus plantarum subgroup (all Firmicutes phylum) were lower in the AN
patients than in the control group of age-matched healthy women. Clostridium coccoides levels
were particularly low in the ANR group [24]. These findings are conflicting in their differing
implications of the effect of AN on Firmicutes abundances.
In the literature, the decrease of Bacteroidetes and increase of Firmicutes has been linked
to obesity in mice [42, 44], although the relationship is less clear in humans [45]. While some
studies on humans found Firmicutes to increase with obesity [46], several other studies did not
show significant differences between the proportions of Bacteroidetes and Firmicutes in obese
and lean subjects [47, 48]. In the Mack et al. study, AN participants and NW participants
demonstrated profound differences in the ratio of abundance of these bacterial phyla. The
relative abundance of Bacteroidetes was significantly lower in AN patients at baseline (T1)
compared to that of NW controls, and the Bacteroidetes abundance was further decreased after
weight gain intervention (T2). Further, the relative abundance of Firmicutes significantly
increased in AN patients between T1 and T2 [22]. Similarly, Morita et al. found that AN patients
had lower counts of Bacteroidetes compared to healthy controls as well, supporting the Mack et
al. finding that Bacteroidetes population decreases in AN. The decrease of Bacteroidetes and
increase of Firmicutes in the Mack et al. study may be due to the increased dietary fat and energy
during treatment, since similar proportions of these phyla have been found in obese mice [42,
44]. Further, Heisen et al. found that obese patients in the LC and VLCD groups had altered
ratios of Bacteroidetes to Firmicutes, while there was no difference found for obese controls
[36]. One study found that a short-term high-calorie diet led to decreased Bacteroidetes and
increased Firmicutes [46]. In mice, decreased Bacteroidetes and increased Firmicutes allowed
increased energy-harvest from the diet [44]. Thus, although the effect of caloric restriction (in
AN and otherwise) on Bacteroidetes and Firmicutes varies as to whether their frequencies
increase or decrease with caloric restriction, caloric restriction clearly has a significant impact on
the ratio of Bacteroidetes and Firmicutes, implying that the impact of these phyla on gut health
warrants further investigation.
Bifidobacteria (of the Actinobacteria phylum) are able to convert carbohydrates to energy
[49]. The Bifidobacteria genus has also been reported to reduce insulin resistance and
inflammation in animal models of metabolic syndrome [50, 51]. In malnourished Bangladeshi
children, no changes in Actinobacteria were observed [52]. However, in fasted humans,
increased bifidobacteria were found [53]. In their study, Morita et al. found that Japanese AN
patients did not exhibit changes in Actinobacteria [24]. In the Mack et al. study, AN patients had
Bifidobacteria in their cores but NW controls did not [22]. Dong et al. found that
Bifidobacterium spp. increased after HPD and NPD [37]. Interestingly, Bifidobacterium spp.
were found to be important therapeutic targets of HPDs [54]. Trinh et al. found that both
Lactobacillus and Bifidobacterium increased in relative abundance in RBW and ABA rats [31].
Caloric restriction studies in obese patients and in food-restricted mice have found increases in
lactobacilli strains, typically associated with beneficial health outcomes, such as increased
longevity and decreased inflammation [55, 56, 57]. Bifidobacterium and Lactobacillus strains are
commonly used as probiotics; thus, their presence during caloric restriction may indicate a
protective mechanism induced by prolonged energy and macronutrient limitations in AN.
In the Mack et al. study, AN patient fecal samples before and after weight gain contained
increased BCFA levels, which are major indicators of microbial protein fermentation [22]. In the
AN participants, the observed increased BCFA concentration likely served as markers for protein
fermentation [58] endogenous support sources (bacterial sections and lysis products, bacterial
cells, etc.); thus, favoring the presences of microbes that act in protein fermentation. Importantly,
both Mack et al. and Morita et al. showed a clear trend for increased BCFA in AN patients (albeit
not statistically significant in the Morita et al. study, likely due to insufficient sample size)
[22,24]. Thus, protein fermentation plays an important role in AN, even after weight gain
treatment. Further, Dong et al. demonstrated that caloric restriction diets impact the gut
microbiome of obese participants in a manner dependent on protein intake. The calorically
restricted HPD induced an increase in intestinal microbial diversity in the participants relative to
the NPD [37]. The mechanism by which the HPD increased microbial richness also had to do
with protein metabolism. In patients on HPD, researchers observed a mechanism that may
involve increased delivery of dietary amino acids to the end of the GI tract for fermentation,
causing intestinal microbes to switch from deriving nitrogen from endogenous sources to
deriving it from dietary-derived protein. Moreover, Heinsen et al. used a VLCD that consisted of
a relatively high amount of protein, and they found that the VLCD led to significant changes in
gut microbiota populations [36]. Thus, the presence of microbes that are implicated in protein
degradation in both obesity and AN may be related to the presence of gut dysbiosis, which is
present in subjects with both conditions.
Increased levels of genera harboring species involved in protein degradation (Clostridium
cluster I (sensu stricto) and cluster XI) were found in AN patients at baseline when compared to
those of controls [22]. In addition to BCFA, phenols and indoles (both carcinogens), ammonia
(mutagen), amines (mutagen precursors), and thiols (cellular toxin) are formed during protein
fermentation [58]. These detrimental metabolites can negatively impact the host gut physiology
and motility, thus, increasing the risk of several UGIS and LGIS [59]. These metabolites can also
contribute to negative psychological outcomes like depression through their interaction with the
gut-brain axis. It is possible that these metabolites could also contribute to a perpetuation of the
somatic consequences of malnutrition in AN patients. Since AN patients in the Mack et al. study
retained GI complaints after weight gain, endogenous protein sources may still be elevated after
weight gain intervention in AN patients despite the increase of diet-derived protein.
Mucins play important lubricative and protective roles, including as protective barriers
against pathogens and chemical and physical damage. During starvation, mucin degradation is a
competitive benefit since other microbiota dependent on dietary nutrients can’t utilize mucin
[60]. However, degrading the mucus layer can increase passage of bacterial products across the
gut wall, causing inflammation. In the literature, the abundance of Verrucomicrobia (primarily
Akkermansia muciniphila) were inversely related to body weight in both mice [61] and humans
[57, 62]. Akkermansia is a mucin-utilizing bacterium. Further, Verrucomicrobia abundance was
increased in fasted hamsters [63], undernourished mice [41], and fasted humans [53]. In their
study, Mack et al. found that several mucin-degrading bacteria (including Verrucomicobia,
Bifidobacteria, and Anaerotruncus) were higher in the nutrient-deprived gut microbiomes of the
AN participants before weight gain when compared to NW controls [22]. Further, higher levels
of Akkermansia were found in the ABA group of rats in the study by Trinh et al. [31]. Similarly,
Dong et al. found that increased abundances of Verrucomicrobia, specifically Akkermansia spp.
were induced by both calorically-restrcited HPD and NPD in obese subjects [37]. However,
Dong et al. did not attribute this to the caloric restriction itself, citing studies in which gut
microbial diversity was not affected by caloric restriction [64, 65].
Dietary fiber serves as a substrate for the gut microbiota, as do proteins, fats, and di- and
monosaccharides [66, 67]. Due to overall decreased macronutrient and energy intake of AN
patients prior to treatment, as such intakes contribute to GI-symptoms, it is likely that dietary
substrates other than fiber are present in the colon in smaller amounts in AN participants than in
healthy subjects. Thus, fiber and microbe-derived proteins and carbohydrates (including
glycoprotein mucins) are the main substrates for the gut microbiota in AN patients. Further,
Dong et al. found that baseline levels of Akkermansia spp. were associated with fiber intake,
suggesting that emphasis on deriving dietary carbohydrate intake from high fiber sources might
have driven the aforementioned microbial increase [37]. These fiber substrates could provide an
advantageous environment for particular microbes (like Akkermansia spp.). Additionally,
Heinsen et al. found that use of a VLCD that was higher in fiber intake than the typical diets of
obese subjects resulted in a significant change in the gut microbiota of obese subjects. Since
constipation-related GI symptoms were common in AN patients in the Mack et al. study and
fiber is a main substrate of the microbiota of AN patients, GI symptoms (infrequent stools,
straining during bowel movement, feeling of incomplete evacuation) may be a result of specific
microbes [68]. Further, in the Mack et al. study, AN patients were subjected to diets high in fat,
fiber, and energy, resulting in slightly improved GI-symptoms [22]. Thus, it is likely that the
increased supply of dietary substrates creates favorable conditions for carbohydrate-metabolizing
microbes, leading to a shift in mucin-degrading taxa. Overall, the presence of GI symptoms
indicated the presence and persistence of gut dysbiosis in AN patients.
In the Mack et al. study, researchers found differences between the gut microbiota of
ANR and ANBP patients [22]. Morita et al. found that Clostridium difficile was only detected in
the ANBP group [24]. This is likely due to the exposure to antimicrobial agents that occurs in
ANBP, which is the most well known risk factor for C. difficile infection. Further, ANBP patients
often have esophageal and gastric abnormalities (UGIS) due to frequent vomiting, which is
consistent with the GI symptoms that were still present in AN patients at T2 in the Mack et al.
study. The persistence of GI symptoms in ANBP patients may indicate a continuation of purging
behaviors that may require further treatment beyond the weight gain program.
Further, the presence of GI symptoms (which are included in the somatic symptoms of
AN) were found to only improve with an increase in weight, as supported by the findings of
Trinh et al. relating to model symptoms in ABA mice [31]. This further supports the finding
from Mack et al. that GI symptoms improved slightly after weight gain intervention.
Another study found that transplantation of Christensenella minuta to germ-free mice
reduced weight gain by altering their gut microbiota [69]. These findings suggest that gut
dysbiosis or a particular microbe found in AN patients’ gut microbiomes may contribute to the
maintenance of emaciation.
In their ABA mice study, Breton et al. found that to meet the energy demands of the host,
gut microbiota could be an alternative source of energy substrates due to the capacity of
microbiota enzymes to catalyze the production of SCFA such as acetate, propionate, and butyrate
[30]. Several studies found low levels of SCFA in AN patients’ feces [19, 22, 24]. This reflects
that a decrease in SCFA-producing microbiota such as Roseburia may be related to caloric
deprivation in AN. Breton et al. found that plasma levels of acetate tended to be higher in ABA
than control mice. In the cross-sectional comparative studies, Morita et al. found decreased acetic
and propionic acid concentrations in AN patients [24]. Meanwhile, Mack et al. did not find
altered levels of acetate and propionate [22]. However, this could be explained by a difference in
the gut microbiota populations of European and Japanese AN patients due to cultural differences
in diet. In the Mack et al. study, decreased levels of Roseburia spp. And Gemminger spp. were
found. Roseburia, an important producer of butyrate, correlated positively for all participants in
the Mack et al. study, indicating its important role in butyrate production and explaining the
decreased butyrate levels in AN patients at T1. Overall, there appears to be a decrease of SCFA
in the GI tracts of AN patients. Whether this potential decrease is the result of caloric restriction
and what its effect may be on microbiota diversity warrants further investigation.
Animal Trials Outcomes
Both of the animal studies analyzed in this review utilized the adjustment of food intake
and physical activity levels, which resulted in an altered gut microbiota [30, 31]. Although
increased physical activity did lead to differences in the gut microbiota, it was not the main
contributor. In the rat study by Trinh et al., the findings indicated that starvation under different
circumstances such as running wheel exposure, reduced bodyweight, or a combination of both,
even without physical activity, resulted in an altered gut microbiota. Chronic starvation, whether
intentional or not, interfered with alpha and beta diversity microbial profiles [31]. This may
suggest that with unintended and general starvation, these extreme measures alone will cause
significant changes in the gut microbiota. The outcomes in the animal trials compared to the
human trials differed, as the diet measures were more extreme with the rodents and the rodents
started at normal weights (in contrast to the obese participants of the human trials). One of the
main differences was the alpha diversity microbial profiles between the rat subjects and human
participants on the HPD. In the rat study, the alpha diversity increased in the food restricted rats
compared to the control groups [36, 37]. The human study by Dong et al. resulted in an initial
decrease in alpha diversity, which was followed by an increase [37]. Between these two studies,
the inconsistency in the results prevents general conclusions from being applied to AN patients.
Animal trials allow for more extreme measures to be implemented with subjects, but it may not
be fully applicable to humans who individually have different starvation patterns. Additionally,
the gastrointestinal function between rodents and humans overall have differences that may have
contributed to the discrepancies within the alpha diversity results. Individually the human and
rodent studies may apply to AN and starvation patients, but not all aspects of the gut microbiota
health.
For the beta diversity results, six genera were of interest as their abundance changed
significantly after induced starvation. Akkermansia, a mucin-utilizing bacterium that increases
intestinal permeability by degrading the protective mucus layer, increased in abundance in
specifically the RWA and ABA groups [31]. Increased abundance of Akkermansia can cause
inflammation and immunological reactions if at a dangerous level. Ruminococcus, a bacteria
related to inflammatory intestinal diseases also increased in response to starvation. Low grade
inflammation is a common finding in patients with AN, which may suggest the danger of
increased abundance of Ruminococcus. Odoribacter also increased in abundance in the
starvation rats, which may be an adaptation response in protecting the strength of the gut wall.
Furthermore, for Lactobacillus and Bifidobacterium, two strains commonly used as probiotics, a
significant increase in relative abundance in RBW and ABA rats may also represent a protective
mechanism induced by prolonged food restriction [31]. These responses may suggest that when
starvation occurs, the body reacts and establishes adaptive mechanisms. This also may be an
opportunity for more studies to be focused on supplementation with Lactobacillus and other
probiotic genera as a treatment intervention for AN patients.
In the mice study by Breton et al., proteomic investigations were performed alongside gut
microbiota measurements [30]. Between the test groups, 16 proteins were overexpressed in the
CT group, 4 in the FTR group, and 26 proteins were different between the FTR and ABA groups.
Of the identified proteins, those that were upregulated in both ABA and FTR mice were from
Clostridiales [30]. Furthemore, increased expression of flagellin, a structural protein, was also
increased in FTR mice. This may suggest that starvation leads to increased metabolic activities
as another coping mechanism, alongside the host adapting to increase energy harvesting and
storage to maintain ATP synthesis.
Strengths
An overall strength of the included research articles was the variation in the types of
starvation patterns studied, specifically comparing caloric restriction, general starvation, and
anorexia nervosa. The examination of the different starvation patterns allowed us to contrast how
each one individually affects the gut microbiota, instead of just focusing on AN. Another
strength was the difference in how each study measured the effect of the starvation pattern. Some
of the studies measured alpha and beta diversity through fecal samples, while others extracted
DNA samples, mRNA levels, or brain weight. This provided insight to how starvation patterns
affect not only the gut microbiota, but also body parts connected to the gut. Incorporating animal
trials was another strength among the research, as they provided alternate data that would not be
possible to observe in humans. Animal trials allow researchers to implement certain diets and
exercise measures that would be inhumane in humans. Both of the rat and mice studies used in
the review further investigated ABA and observed whether or not intense physical activity could
alter the gut microbiota. Scientists could alter physical activity levels or food consumption of the
rats and mice quickly without any conflict. Another strength in the studies was the wide range of
participants’ age, as the youngest participants were 23.7± 8-23.8 ± 6.7 years in the study by Mack
et al., and the oldest participants were 55.7±.4-55.9±10.1 years in the study by Dong et al. [22,
37].
Limitations
A common limitation among the studies was a low sample size, as five of the six studies
had less than 100 participants [24, 30, 31, 36, 37]. With newer topics in the nutrition field, it’s
important that any research conducted leaves little room for error or false conclusions among
variables. Another limitation was the gender of the participants, as each study either mimicked
female AN/starvation patients or focused on male subjects. Although most AN patients are
female, the lack of gender diversity in the studies renders their conclusions ungeneralizable
beyond each study’s individual populations. A limitation of the rat study, in particular, was that
only the amount of food was changed, not the food composition/type. Patients with AN often
consume vegetarian/vegan diets, restrict fat or carbohydrate intake, or follow other restrictive
dietary patterns [31]. To mimic other common diet patterns amongst AN patients would have
allowed for more accurate representations of what AN patients typically eat. The findings from
the animal studies in this review may not be generalizable to humans. Another limitation to the
study by Heinsen et al. was that many of the subjects were on medication and/or had other
pre-existing health conditions such as diabetes and hyperuricemia [36]. This may have resulted
in the independent variable displaying a more significant effect on the participants than it would
otherwise. The pre-existing conditions may have confounded the results. Another limitation was
the relative lack of participants in the non-adolescent age range (although some studies included
adolescent participants, the average ages were considerably older). Although the studies
provided a great range of ages, especially in the middle age group, they involved non-adolescent
participants. Because most AN patients are adolescent females ages 15-24, additional studies
with participants in this age range would have provided more reflective results of those primarily
affected by AN [13].
Conclusion and Implications for Further Study:
The gut microbiome is related to a plethora of important health outcomes. Gut
microbiome diversity is implicated in digestion control and immune function. The gut microbiota
has an impact on many diseases, including GI-diseases (IBD, IBS) and neurodegenerative
diseases (AD, PD, MS). Due to the broad spectrum of effects the gut microbiota has on health, it
is difficult to quantify the monetary, physical, and emotional burdens that gut dysbiosis has on
the population, though it can be assumed to be quite significant. The purpose of this review was
to investigate what the effect of AN and other forms of caloric deprivation had on the gut
microbiome. Overall, the review articles indicated mixed results as to whether caloric restriction
resulted in an increase or decrease in gut microbiota diversity. The four starvation studies found
that once caloric restriction began, alpha diversity increased, generally indicating that caloric
restriction did not result in gut dysbiosis. In contrast, the cross-sectional AN studies showed that,
while there were few differences between the gut microbiota of the AN patients and the healthy
control subjects, the gut microbiota diversity of AN patients was similar to or lower than that of
controls, suggesting that long term caloric restriction in AN may result in gut dysbiosis. Since
increased microbial diversity is indicated with caloric restriction, except in the case of AN, there
may be other factors occurring in AN causing gut dysbiosis. The identity of these potential
factors warrants further study. Despite their differing microbiota diversity results, both the
starvation (obesity and rodent model studies) and AN cross-sectional studies indicated that
caloric restriction resulted in changed ratios of Firmicutes and Bacteroidetes in the microbiota.
Further studies should investigate the effect of caloric restriction in AN and in treatment of other
conditions on the ratio of Firmicutes and Bacteroidetes. Such an investigation could inform
development of novel treatments for AN and starvation. Clearly, given the importance of the gut
microbiota in many health outcomes, particularly AN (which has the highest mortality rate of
any mental illness) further investigation into the impact of AN on gut microbiota diversity is
warranted.
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Starvation and the Gut Microbiome 1

Sarah Tu, Jennifer Zielke, Caitlyn Lew

California Polytechnic State University, San Luis Obispo, CA

FSN 281 Section 1

June 29, 2021

Anorexia nervosa (AN) is an eating disorder with a progression characterized by

high-density lipoprotein (HDL) cholesterol, AN is considered to have metabo-psychiatric origins

AN leads to severe endocrine and somatic consequences including increased cortisol

equivalent to those of schizophrenia and depression patients [17].

(severe protein-energy malnutrition) induced altered protein and carbohydrate metabolism in

mice [27, 28].

similar symptoms [2, 29]. Because AN is foremost a psychological disorder, it is important to

the differences between them may impact treatment of malnutrition.

information and an analysis of six original research articles including cross-sectional

June 24 and July 1, 2021.

Interventional Trials on Obese Subjects

Figure 1: Bray-Curtis dissimilarity capscale showing the explained variance at 0-months

decreased from baseline.

Trials on Animal Subjects

In a cross-sectional, randomized study by Breton et al., researchers investigated whether

0.05). Increased levels of phosphoglycerate kinase, the ATP-producing glycolytic enzyme

Lachnospiraceae were increased in the starvation groups.

Cross-Sectional Studies Comparing AN Patients and Control

In a cross-sectional comparative study, Morita et al. compared the fecal microbiota of

based on rRNA-target RT-quantitative PCR technology. The participants were Japanese AN

albumin, blood urea, nitrogen, electrolytes, creatinine, aspartate aminotransferase, alanine

coccoides group, Clostridium leptum subgroup, Bacteroides fragilis group, Bifidobacterium,

Atopobium cluster, Prevotella, Enterobacteriaceae, Enterococcus, Staphylococcus,

Streptococcus, Clostridium difficile, Clostridium perfringens, and total Lactobacillus.

represent the proportion of variance. Data from [24].

their fecal samples than the control group did.

In their cross-sectional comparative study, Mack et al. comprehensively investigated the

As controls, 55 healthy female NW participants were selected. The NW participants were

sequences in Operational Taxonomic Units (OTUs) at 97% similarity. To evaluate differences in

determined using db-RDA analysis of BC distances of samples from AN patients at T1 and NW

determined using the Wilcoxon signed-rank test and Mann-Whitney-U-test.

Compared to NW controls, AN patients had higher levels of LGIS and particularly

phyla were Bacteroidetes, Firmicutes, Actinobacteria, Proteobacteria, and Verrucomicrobia. The

relative abundance of Bacteroidetes was significantly lower in AN patients at T1 than in NW

Firmicutes was higher than that of the NW controls.

In AN patients at both T1 and T2, Actinobacteria levels were high compared to NW

significantly increased in ANT2 patients compared to that of NW controls.

Bray-Curtis distances. In AN patients, the intra-individual dissimilarity in microbial composition

Results Summary Table

Study # Age Independ Dependent Study Design Results

Heinsen 18 Mean: VLCD Diversity in Randomized Increase in

T. Dong 80 NPD: High Diversity of Randomized Initial decrease

J. 32 N/A Food Pellets and Cross-sectional, Sixteen

S. Trinh 49 4 Food ɑ-diversity Randomized Food restricted

activity RBW and

C. 46 (25 AN Control AN Diversity of Cross-sectional AN patients

Acetic acid and

I. Mack 110 (55 NW: AN Diversity of Cross-sectional Bacteroidetes

subjects, randomized interventional trials on animal models, and cross-sectional studies

several groups of microbiota [22].

microbiome of an AN patient that had maintained their caloric-restrictive lifestyle for an

diversity in the patients [24].

improve microbiota diversity, it does not return diversity to a pre-AN state.

AN patients causes them to continue their behavior.

Typically, lower bacterial diversity is considered a detriment to intestinal health. It has

restriction leading to decreased microbiota diversity, including in neonatal mice subjected to

The combination of these factors allowed the normal-appearing alpha-diversity in AN patients.

Wiley & Sons, Inc; 2017.