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Fenugreek (Trigonella foenum-graecum) is an annual herbaceous plant and a staple of traditional health remedies for metabolic conditions including high cholesterol and diabetes. While the mechanisms of the beneficial actions of fenugreek remain unknown, a role for intestinal microbiota in metabolic homeostasis is likely. To determine if fenugreek utilizes intestinal bacteria to offset the adverse effects of high fat diets, C57BL/6J mice were fed control/low fat (CD) or high fat (HFD) diets each supplemented with or without 2% (w/w) fenugreek for 16 weeks. The effects of fenugreek and HFD on gut microbiota were comprehensively mapped and then statistically assessed in relation to effects on metrics of body weight, hyperlipidemia, and glucose tolerance. 16S metagenomic analyses revealed robust and significant effects of fenugreek on gut microbiota, with alterations in both alpha and beta diversity as well as taxonomic redistribution under both CD and HFD conditions. As previously reported, fenugreek attenuated HFD-induced hyperlipidemia and stabilized glucose tolerance without affecting body weight. Finally, fenugreek specifically reversed the dysbiotic effects of HFD on numerous taxa in a manner tightly correlated with overall metabolic function. Collectively, these data reinforce the essential link between gut microbiota and metabolic syndrome and suggest that the preservation of healthy populations of gut microbiota participates in the beneficial properties of fenugreek in the context of modern Western-style diets.
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Fenugreek Counters the Eects of
High Fat Diet on Gut Microbiota in
Mice: Links to Metabolic Benet
Annadora J. Bruce-Keller1*, Allison J. Richard1, Sun-Ok Fernandez-Kim1, David M. Ribnicky2,
J. Michael Salbaum1, Susan Newman1, Richard Carmouche1 & Jacqueline M. Stephens1
Fenugreek (Trigonella foenum-graecum) is an annual herbaceous plant and a staple of traditional health
remedies for metabolic conditions including high cholesterol and diabetes. While the mechanisms
of the benecial actions of fenugreek remain unknown, a role for intestinal microbiota in metabolic
homeostasis is likely. To determine if fenugreek utilizes intestinal bacteria to oset the adverse
eects of high fat diets, C57BL/6J mice were fed control/low fat (CD) or high fat (HFD) diets each
supplemented with or without 2% (w/w) fenugreek for 16 weeks. The eects of fenugreek and HFD on
gut microbiota were comprehensively mapped and then statistically assessed in relation to eects on
metrics of body weight, hyperlipidemia, and glucose tolerance. 16S metagenomic analyses revealed
robust and signicant eects of fenugreek on gut microbiota, with alterations in both alpha and beta
diversity as well as taxonomic redistribution under both CD and HFD conditions. As previously reported,
fenugreek attenuated HFD-induced hyperlipidemia and stabilized glucose tolerance without aecting
body weight. Finally, fenugreek specically reversed the dysbiotic eects of HFD on numerous taxa
in a manner tightly correlated with overall metabolic function. Collectively, these data reinforce the
essential link between gut microbiota and metabolic syndrome and suggest that the preservation
of healthy populations of gut microbiota participates in the benecial properties of fenugreek in the
context of modern Western-style diets.
Obesity linked to Western-style diets is the prototypical ailment of the modern era. Obesity currently aects more
than 35% of Americans1; and in addition to ties with type 2 diabetes and cardiovascular disease, obesity increases
the risk of all-cause mortality and exacerbates anxiety and depression25. While search for eective obesity treat-
ments has become a priority in biomedical research, available pharmacological options for obesity are under-
mined by issues related to toxicity and o-target side6. Herbal medicine or phytotherapy has long been a source
of traditional medicinal remedies, and indeed, interest in generally regarded as safe (GRAS) plant materials for
the clinical treatment of obesity is growing (reviewed in7,8). Fenugreek (Trigonella foenum-graecum) is an annual
herbaceous plant and a staple of traditional health remedies to treat hyperlipidemia and diabetes912, as well as
mood disorders13. Laboratory studies demonstrate protective eects of fenugreek on diabetes1418, and suggest
that potential mechanisms might include inhibition of intestinal glucose absorption1416, delayed gastric empty-
ing15, and/or insulinotropic activity19,20,17,18. Protective eects of fenugreek on cholesterol and hyperlipidemia21
might be based on modulation of hepatic steatosis2226, inammation2628, and/or oxidative stress secondary to
diabetes2932. While the exact mechanisms whereby fenugreek or its constituents confers metabolic resiliency are
unknown, data show that fenugreek administration can also modulate intestinal microbiota, which can in turn
impact metabolic physiology33,34.
A remarkably mutualistic relationship exists between gut microbiota and their mammalian hosts, with micro-
biota providing protection against ingested pathogens, neutralizing carcinogens, and metabolizing otherwise
inaccessible lipids and polysaccharides into potent bioactive metabolites35. Sequencing data show that mod-
ern high fat/calorie diets can disrupt gut microbiota, reducing bacterial diversity and upsetting the balance of
pathogenic and commensal bacteria36. Data from our lab and others show that such diet-induced gut dysbiosis
is sucient to impair both metabolic and neurologic function37,38, suggesting that preservation of healthy gut
microbiota could oset the pathophysiologic eects of high fat diets39. As fenugreek has indeed been shown
1Pennington Biomedical Research Center, Louisiana State University System, Baton Rouge, LA, 70808, USA.
2Department of Plant Biology, Rutgers University, New Brunswick, NJ, 08901, USA. *email: annadora.bruce-keller@
pbrc.edu
OPEN
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to modulate intestinal bacteria in several models33,34,40, studies were designed to determine if fenugreek could
oset the eects of a high fat diet on gut dysbiosis, and to establish the relationship of fenugreek-shaped gut
microbiota to clinically relevant metrics of metabolic function. To this end, data from our previously published
study on the eects of fenugreek on mice given a high fat diet were extended to include sequencing and statistical
assessment of gut microbiota. As reported in our previous study, high fat or nutritionally matched low fat diets
supplemented with ground fenugreek seeds (2% w/w) were administered to male C57BL/6J mice for 16 weeks,
and the metabolic eects of the various diets on adiposity, glycemic control, and hyperlipidemia were quantied41.
Metagenomic sequencing of fecal microbiota collected from mice was conducted, and diet-related changes in gut
microbiota were statistically analyzed in relation to established metrics of metabolic function.
Results
Fenugreek improves glucose tolerance and dyslipidemia in mice given high fat diet. Data in
this manuscript is built on initial publication of the eects of whole fenugreek seed supplementation (2% w/w)
on overall metabolic function in the context of a 16-week trial of high fat diet consumption41, and thus previously
published data are only summarized in this report. Briey, data show that fenugreek supplementation increased
HDL and decreased LDL cholesterol levels in high fat fed-mice (Table1). Furthermore, fenugreek signicantly
improved glucose tolerance (as measured 40 minutes aer oral glucose loading), but did not aect HFD-induced
changes in total cholesterol, body weight, amount of body fat, or fasting blood glucose (Table1). Fenugreek
administration did not cause changes in food intake41.
Fenugreek and high fat diet exert pronounced effects on gut microbial composition. The
impact of fenugreek on intestinal microbiota was determined by 16S sequencing of fecal samples isolated from
mice at euthanasia as described in Methods. Initial weighted and unweighted Unifrac phylogenetic analyses
reveal that the microbiomes of fenugreek-fed mice were signicantly dierent from non-fenugreek mice under
both control diet and high fat-fed conditions (Table2). Indeed, the magnitude of the eects of fenugreek were
similar in that of the high fat dies as compared to control diet (Table2). ese robust shis in beta-diversity were
also apparent on principal component analysis plots generated from normalized read count data (Fig.1). Finally,
CD CD/FG HFD HFD/FG
Total Cholesterol
(mg/dl) 148.9 ± 45.7 136.2 ± 31.7 255.4 ± 32.9*** 245.7 ± 26.3
LDL Cholesterol
(mg/dl) 9.62 ± 2.6 8.48 ± 2.1 17.98 ± 4.7*** 13.83 ± 4.3#
HDL Cholesterol
(%TC) 45.15 ± 12.7 44.86 ± 5.8 28.28 ± 3.3*** 33.3 ± 5.1#
Body Weight (gr) 31.78 ± 3.2 31.57 ± 2.6 48.73 ± 2.7*** 50.03 ± 2.2
Body Fat (gr) 5.15 ± 1.8 5.04 ± 1.2 16.61 ± 1.3*** 16.86 ± 1.3
Fasting Blood
Glucose (mg/dl) 153.4 ± 16.8 149.4 ± 23.4 211.1 ± 16.8*** 219.1 ± 21.1
Glucose Tolerance
(40 min) 234.0 ± 49.3 213.5 ± 23.3 386.1 ± 89.9*** 311.6 ± 75.8#
Table 1. Summary of fenugreek-induced metabolic resiliency: decreased hyperlipidemia and improved glucose
tolerance. Adult male C57Bl/6 mice were given high fat (HFD) or nutritionally matched control diet (CD) with
or without fenugreek (FG; 2% w/w), and subject to measures of metabolic function as described in Methods.
Statistically signicant dierences in metabolic parameters in HFD-fed mice as compared to CD-fed mice are
mice are noted by ***(p < 0.001), while signicant changes in mice given HFD/FG as compared to HFD-fed
mice are noted by #(p < 0.05). Adapted from previously published data41.
Comparison Score P value
Weighted Unifrac
CD vs CD/FG 0.65012 <0.001***
HFD vs HFD/
FG 0.61915 <0.001***
CD vs HFD 0.796987 <0.001***
Unweighted Unif rac
CD vs CD/FG 0.90777 <0.001***
HFD vs HFD/
FG 0.885073 0.002009**
CD vs HFD 0.952579 0.001009**
Table 2. Dierences in microbiota community composition in mice with CD- and HFD-shaped microbiota
with and without fenugreek. Operational taxonomical units (OTU) were identied based on sequence
clustering as described in Methods, and generation of a read count table was performed with the soware
package ‘usearch. Statistical tests for dierential representation were performed with tools incorporated in
‘mothur’, and statistically signicant dierences in microbiota community composition between groups were
detected using both weighted and unweighted Unifrac phylogenetic analysis tools.
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data show that fenugreek also signicantly increased alpha diversity (Shannon metrics) in both control diet and
high fat-fed mice (Fig.2).
Fenugreek can correct the dysbiotic effects of high fat diet on intestinal microbial popu-
lations. To assess the impact of fenugreek and HFD on gut microbial composition in greater statistical
detail, a dierential analysis of count data was conducted using DESeq. 2. Specically, the specic individual
operational taxonomic units (OTUs) whose relative representation was signicantly (p < 0.05 adjusted with
Benjamini-Hochberg correction) changed by high fat diet were identied using DESeq. 2. ese analyses revealed
that out of 410 Core OTUs (identied in all mice), the relative representation of 147 individual OTUs was sig-
nicantly dierent in HFD-fed mice as compared to CD-fed mice (Fig.3); with 57 increased and 90 decreased,
respectively, by HFD. A similar DESeq. 2 analysis of these 147 “HFD-transformed” taxa was conducted to identify
those that were signicantly aected by fenugreek such that the direction of the change induced by high fat diet
was reversed. is ivvestigation of “fenugreek-corrected” taxa revealed that fenugreek corrected the eects of
HFD on 50 of these OTUs by reducing the representation of 27 HFD-increased OTUs and augmenting the rep-
resentation of 23 OTUs reduced by HFD (Fig.3). ese analyses reect the robust eect of both fenugreek and
HFD on gut microbiota, and show that fenugreek is able to signicantly correct much (greater than 34%) of the
dysbiotic eects of HFD.
Representation of Fenugreek-corrected taxa largely predicts overall metabolic function. In
the nal set of analyses, the 50 fenugreek-corrected taxa whose representation was skewed in one direction by
HFD but in the opposite direction by fenugreek was examined in relation to metabolic function. Specically, to
determine if the relative representation of fenugreek-corrected taxa could be used to predict metabolic resiliency,
the statistical relationship of fenugreek-corrected taxa representation to the metrics of metabolic function listed in
Table1 was determined. To this end, a matrix was built containing OTU count data for all 50 fenugreek-corrected
Figure 1. Fenugreek changes intestinal microbial populations in mice. Fecal microbiome populations from CD,
CD/FG, HFD, and HFD/FG mice were analyzed using 16S rRNA sequencing, and multi-dimensional scaled
principal coordinate analysis were used to visualize UniFrac distances of fecal samples from individual recipient
mice. Samples from CD, CD/FG, HFD, and HFD/FG mice are depicted as blue, purple, red, and green symbols,
respectively.
Figure 2. Fenugreek increases overall intestinal microbial diversity. Fecal microbiome populations from CD,
CD/FG, HFD, and HFD/FG mice were analyzed using 16S rRNA sequencing, and box plots were generated to
depict dierences in Shannon α-diversity. Data show that mice supplemented with 2% fenugreek in their feed
exhibited a statistically signicant (**p < 0.01) increase in α-diversity compared to mice given CD or HFD
alone.
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taxa along with all metabolic data depicted in Table1, including those indices not aected by fenugreek. Pairwise
Pearson correlations indicate that the relative expression of many of these 50 OTUs were highly predictive of met-
abolic function. For example, of the 23 taxa decreased by HFD but increased with fenugreek supplementation, 8
taxa (all in the fermicutes phylum) signicantly correlated with at least 1 metric of metabolic function (Table3,
see Supplementary Table1 for additional details (log2FC and Pearson r values) on HFD-decreased, fenugreek
corrected taxa). Likewise, of the 27 taxa increased by HFD but decreased with fenugreek supplementation, 20
taxa signicantly correlated with selected metrics of metabolic function (Table4, see Supplementary Table2 for
additional details (log2FC and Pearson r values) on HFD-increased, fenugreek corrected taxa). It is important
to note that the representation of fenugreek-corrected taxa correlated frequently with aspects of metabolic func-
tion (e.g., body weight, body fat, total cholesterol, fasting blood glucose) that were not signicantly improved in
fenugreek-treated mice.
Discussion
While benecial eects of fenugreek on hyperlipidemia and hyperglycemia have been widely reported, the mech-
anisms of fenugreek-mediated actions on metabolic function are unknown. Here we demonstrate the robust
eects of fenugreek on gut microbiota, and describe a novel combination of statistical analyses including Unifrac,
iterative DESeq. 2, and pairwise correlation matrices to generate insight into the role of intestinal microbial
changes in the protective eects of fenugreek. Specically, sequencing analyses reveal that fenugreek signicantly
increased overall microbiome diversity in mice, and specically reversed the actions of high dietary fat on key
intestinal taxa. Furthermore, the representation of fenugreek-corrected taxa signicantly correlated with meta-
bolic function, including changes in body weight and composition, glucose regulation, and hyperlipidemia.ese
ndings are in agreement with the extensive body of literature documenting the ability of high fat diet to reduce
bacterial diversity and disrupt the balance of pathogenic/commensal bacteria within the intestine36,42,38,43. As data
from our lab and others show that this pattern of gut dysbiosis is sucient to impair both metabolic and neuro-
logic function37,4448, data in this paper suggest that the reported eects of fenugreek on gut microbiota33,34,40,49,
may be fundamental to its benecial properties. In light of the stubborn prevalence of obesity and the pervasive
accessibility of unhealthy diets, it is both clinically signicant and generally promising that these data suggest
that partial reversal of gut dysbiosis and metabolic impairment can be achieved with botanical supplementation
within the context of unhealthy, Western-style diets.
While the association of intestinal dysbiosis with metabolic disease is well established4548 causal relationships
have not been identied and it is not understood how microbiome constituents impact metabolic resilience/vul-
nerability. ree major phyla are the most abundant in the human distal intestine: Bacteroidetes (gram-negative),
Firmicutes (gram-positive), and Actinobacteria (gram-positive). Early studies suggested that obesity causes
reductions in Bacteroidetes and increases in Firmicutes38,43 and indeed further studies indicate that these changes
are reversed with weight loss50,51. While these data suggest that the balance between these phyla might broadly
impact host physiology, this binary distinction does not always occur52,53 and may be too simple to faithfully
reect the complexity of diet-induced changes to the gut microbiome5456. In the present study as well as our
previous studies54,57, dierences between control and high fat groups did not manifest as phylum-level shis
but rather dierential representation within taxa, particularly Firmicutes. Indeed, the majority of taxa included
in Tables3 and 4 arise from the Clostridium class (Clostridium cluster XIVa) or the Clostridium leptum group
(Clostridium cluster IV). is is notable, as divergent shis in the representation of Clostridia have been reported
in other pathophysiological conditions. For example, while overall Clostridium representation generally increases
with age, Clostridium XIVa clusters have been shown to be signicantly reduced in the elderly58. e bacteria
in Clostridium XIVa play major roles in the fermentation of carbohydrates within the gut59, and the major end
products of hind-gut fermentation are short-chain fatty acids (SCFAs). Further, Fermicutes produce primarily
butyrate as their metabolic end product60, and butyrate is the main source of nutrition for gut epithelium cells61.
Depletion of butyrate is associated with impaired intestinal barrier integrity62, and loss of intestinal barrier func-
tion is in turn associated with a growing number of inammatory disease states diseases, including obesity as well
as autoimmune diseases and cancer (reviewed in63,64). While butyrate was not directly measured in the present
Figure 3. Graphical representation of fenugreek’s ability to reverse HFD-induced changes in individual
intestinal microbiota. e ability of high fat diet to signicantly alter the representation of individual taxa was
assessed as described in Methods showing that out of 410 Core OTUs, 57 were signicantly increased and 90
signicantly decreased by HFD. Fenugreek supplementation signicantly reversed the eects of HFD on 50 of
these OTUs by reducing the representation of 27 HFD-increased OTUs and bolstering the representation of 23
OTUs reduced by HFD.
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study, decreased levels of butyrate and other SCFA are widely reported in the context of obesity while high-ber
plant products are known to increase colonic fermentation and the generation of SCFA65.
Our data indicate that fenugreek is particularly eective against hyperlipidemia, which is in keeping with
results of both experimental and clinical studies14,22,30,66. While there are several mechanisms whereby changes
in gut microbiota could mediate the eects of fenugreek on serum lipids, most ultimately impact the absorp-
tion of dietary fat. For example, intestinal microbiota alter the metabolism of diet-derived long-chain fatty acids
such as conjugated linoleic acid, modulating absorption67. Gut microbiota can also moderate cholesterolemia
Correlation (Pearson) of HFD-Decreased, Fenugreek-Corrected OTU’s to Metrics of Metabolic Function
Individual OTUs Decreased by High Fat Diet (HF) Corrected by FG Total
Chol. LDL
Chol. HDLChol. Body
Wei g ht Bod y Fat Fasting
Glucose Glucose
Tolerance
Firmicutes/Clostridia/Clostridiales/Lachnospiraceae/Clostridium_XlVa/Species1 ns ns ns ns 0.0014 ns 0.0001
Firmicutes/Clostridia/Clostridiales/Lachnospiraceae/Clostridium_XlVa/Species2 ns ns ns 0.0027 0.0025 ns ns
Firmicutes/Clostridia/Clostridiales/Lachnospiraceae/Clostridium_XlVa/Species3 0.0001 0.0034 0.0011 ns ns 0.0006 0.0001
Firmicutes/Clostridia/Clostridiales/Ruminococcaceae/Flavonifractor 1.2E-05 0.0005 0.0001 1.6E-06 2.1E-06 2.2E-05 1.9E-05
Firmicutes/Erysipelotrichia/Erysipelotrichales/Erysipelotrichaceae/Turicibacter 0.0004 ns 0.0024 ns ns ns 0.0015
Firmicutes/Clostridia/Clostridiales/Ruminococcaceae/Oscillibacter 0.0002 ns 1.3E-05 ns 4.0E-05 0.0002 4.2E-05
Firmicutes/Clostridia/Clostridiales/Ruminococcaceae/Intestinimonas 0.0009 0.0021 0.0026 ns ns 0.0004 0.0017
Firmicutes/Clostridia/Clostridiales/Lachnospiraceae/Acetatifactor/Species 1 ns ns ns ns ns ns 0.0012
Table 3. OTUs that predicts metabolic decline in high fat-fed mice. Individual OTUs in which fenugreek
administration reversed high fat diet-induced decreases in representation were correlated against measures of
hyperlipidemia. Data are p values of Pearson correlation with total cholesterol (mg/dl), low-density lipoprotein
(LDL Chol.; mg/dl), high-density lipoprotein (HDL Chol.; %TC), body weight (grams), body fat (grams),
fasting blood glucose (mg/dl), and glucose tolerance (blood glucose levels 40 minutes aer oral loading). See
Supplementary Table1 for additional details (log2FC and Pearson r values) on HFD-decreased, fenugreek
corrected taxa.
Correlation (Pearson) of HFD-Increased, Fenugreek-Corrected OTU’s to Metrics of Metabolic Function
Individual OTUsDecreased by High Fat Diet (HF) Corrected by FG Total
Chol. LDL
Chol. HDL
Chol. Body
Wei g ht Bod y Fat Fasting
Glucose Glucose
Tolerance
Firmicutes/Clostridia/Clostridiales/Lachnospiraceae/Clostridium_XlVa/Species 4 ns 4.9E-05 ns ns ns ns ns
Firmicutes/ClostridiaClostridiales/Ruminococcaceae/Anaerotruncus 0.0004 1.3E-08 ns ns 0.0016 ns 4.6E-06
Firmicutes/Clostridia/Clostridiales/Lachnospiraceae/Clostridium_XlVa/Species 5 ns 5.8E-05 0.0024 ns ns ns ns
Firmicutes/Clostridia/Clostridiales/Lachnospiraceae/Clostridium_XlVa/Species 6 0.0002 4.9E-09 0.0051 ns ns ns 0.0002
Bacteroidetes/Bacteroidia/Bacteroidales/Porphyromonadaceae/Barnesiella/Species 1 9.1E-05 4.1E-10 ns 0.0009 0.0009 0.0024 1.6E-06
Bacteroidetes/Bacteroidia/Bacteroidales/Porphyromonadaceae/Barnesiella/Species 2 0.0005 4.2E-09 ns ns ns ns 8.8E-06
Firmicutes/Clostridia/Clostridiales/Lachnospiraceae/Clostridium_XlVa/Species 7 8.1E-07 5.7E-10 4.8E-05 ns ns ns 0.0001
Firmicutes/Clostridia/Clostridiales/Lachnospiraceae/Clostridium_XlVa/Species 8 0.0001 0.0002 0.0030 ns 1.2E-05 0.0011 1.3E-05
Firmicutes/Clostridia/Clostridiales/Lachnospiraceae/Clostridium_XlVa/Species 9 4.1E-06 5.9E-08 0.0007 ns 1.2E-06 4.1E05 ns
Firmicutes/Bacilli/Lactobacillales/Streptococcaceae/Streptococcus ns ns ns ns ns ns 0.0007
Firmicutes/Clostridia/Clostridiales/Lachnospiraceae/Clostridium_XlVa/Species 10 0.0009 2.1E-05 ns ns 0.0002 ns 5.0E-06
Firmicutes/Bacilli/Lactobacillales/Lactobacillaceae/Lactobacil lus/Species 1 4.2E-06 ns ns 7.1E-08 4.3E-09 5.6E07 1.1E-06
Firmicutes/Bacilli/Lactobacillales/Lactobacillaceae/Lactobacil lus/Species 2 1.3E-06 0.0001 ns 2.9E-08 2.1E-09 2.9E-07 5.2E-07
Actinobacteria/Actinobacteria/Coriobacteridae/Coriobacteriales/Coriobacterineae ns ns ns ns ns ns 0.0033
Firmicutes/Clostridia/Clostridiales/Lachnospiraceae/Roseburia 6.0E-05 5.4E-07 0.0004 ns ns ns ns
Firmicutes/Clostridia/Clostridiales/Lachnospiraceae/Clostridium_XlVa/Species 11 ns ns ns 0.0030 0.0016 ns 0.0015
Firmicutes/Clostridia/Clostridiales/Clostridiales_Incertae_Sedis_XI/Dethiosulfatibacter 0.0039 ns ns 0.0004 ns ns
Bacteroidetes/Bacteroidia/Bacteroidales/Porphyromonadaceae/Barnesiella/Species 3 ns 1.4E-05 ns ns ns ns 0.0040
Firmicutes/Clostridia/Clostridiales/Lachnospiraceae/Acetatifactor/Species 2 0.0003 0.0002 ns ns 1.6E-05 ns ns
Firmicutes/Clostridia/Clostridiales/Lachnospiraceae/Clostridium_XlVa/Species 12 5.2E-05 2.2E-07 0.0003 ns ns ns ns
Table 4. OTUs that predicts metabolic decline in high fat-fed mice. Individual OTUs in which fenugreek
administration reversed high fat diet-induced increases in representation were correlated against measures of
hyperlipidemia. Data are correlation coecients (Pearson r) and p values of correlation with total cholesterol
(mg/dl), low-density lipoprotein (LDL Chol.; mg/dl), high-density lipoprotein (HDL Chol.; %TC), body weight
(grams), body fat (grams), fasting blood glucose (mg/dl), and glucose tolerance (blood glucose levels 40 minutes
aer oral loading). See Supplementary Table2 for additional details (log2FC and Pearson r values) on HFD-
increased, fenugreek corrected taxa.
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by regulating cholesterol conversion into coprostanol68. Dietary cholesterol is largely incorporated into chylo-
microns for absorption in the small intestine. However, signicant quantities of cholesterol (as much as 1 gram
per day) escape proximal absorption to enter the colon to be either be excreted or absorbed. Microbial-based
metabolism of cholesterol into coprostanone/coprostanol reduces blood cholesterol by increasing fecal choles-
terol excretion69,70. Interestingly, recent data suggest that taxa arising from Lachnospiraceae and Runinococcacea
families of the phylum Fermicutes are uniquely associated with high coprostanol generation in healthy humans71,
while other studies likewise link these gut microbiota to variation in blood lipid levels independently of age, sex,
and host genetics72. It is important to note that seven of the eight of the taxa listed in Table3 are members of
Lachnospiraceae and Runinococcacea families, suggesting that reversal of HFD-induced decreases in these key
taxa by fenugreek could remediate hyperlipidemia by promoting fecal fat excretion. In further support of this
scenario, published data suggest that fenugreek supplementation can dose-dependently increase fecal excretion of
cholesterol from rats given high fat/high calorie diets73. Collectively, these data raise the possibility that increased
representation of coprostanoligenic taxa arising from Lachnospiraceae and Runinococcacea families could par-
ticipate in the lipid-lowering eects of fenugreek, and further suggest that identication and analysis such strains
could lead to improved understanding and management of hypocholesteremia.
Transformation and metabolism of bile acids is another key pathway whereby fenugreek-shaped intestinal
bacteria could impact serum lipids (reviewed in74). Indeed, fenugreek has been reported to inhibit the intestinal
absorption of primary and secondary bile acids75, and to increase bile acid excretion into feces30. While the bulk of
bile acids released into the intestine are eciently absorbed and recycled back to the liver, perhaps 5% of the total
bile acid pool progresses into the colon. Bile acids reaching the colon are subject to several microbial-mediated
reactions including transformation into secondary bile acids by dihydroxylation, and deconjugation by bile
salt hydrolases. While data indicate that bile salt hydrolases are a pervasive microbial adaptation to the human
gut environment with enrichment in major genera including Bacteroides, Clostridium, Lactobacillus, and
Eubacterium76, probiotics with bile salt hydrolytic activity can lower serum cholesterol77,78. With regard to more
broad metabolic benets, secondary bile acids generated by microbial metabolism are potent ligands of the
G-protein coupled receptor TGR5, the activation of which triggers release of GLP-1 and insulin, thereby modu-
lating host glucose tolerance and energy expenditure79.
While this study is in keeping with an extensive body of literature on the benecial eects of fenugreek, the use
of whole seed supplementation precludes identication of the bioactive constituent(s) mediating changes to gut
microbiota. Furthermore, only male mice were used, so sex-based dierences in the eects of fenugreek or the
relation of such to metabolic function cannot be resolved. is point is especially signicant as female C57BL/6J
mice are generally considered resistant to diet induced obesity80,81. Notwithstanding these limitations, these data
indicate that fenugreek supplementation can stabilize metabolic function within the context of high fat consump-
tion. Indeed, fenugreek did not aect food intake or alleviate diet-induced obesity, but rather was able to bolster
resistance of the obese mice to hyperlipidemia and glucose intolerance. We use the term “metabolic resiliency” to
describe this ability to preserve, at least in part, a healthy metabolic phenotype in the context of powerful external
stressors – in this case sustained consumption of a high fat diet and the obese state. is is a signicant nding,
as while the components of a healthy lifestyle are generally well known, numerous societal factors including
poverty, food deserts, irregular/sedentary work schedules combine to hinder a consistently healthy lifestyle for
most Americans. us, use of fenugreek and/or other strategies to maintain a healthy population of intestinal
microbes in the context of a high fat diet could foster metabolic resilience even when diets/lifestyles are not opti-
mal. Indeed, while fenugreek conferred protection against hyperlipidemia and glucose intolerance, it is important
to note that representation of specic fenugreek-corrected taxa correlated frequently with aspects of metabolic
function (e.g., body weight, body fat, total cholesterol, fasting blood glucose) that were not signicantly improved
in fenugreek-treated mice (see Tables3 and 4). While correlation does not equal causation, these data clearly
illustrate the very close relationship of individual microbes and microbial balance with metabolic function, and
raise the possibility that strategic manipulation of key intestinal taxa could result in a more complete reversal of
the adverse action of high fat diet. us, the action of fenugreek could be possibly optimized by dose, preparation,
or combination with additional factors (probiotics, etc) to have a greater eect on key microbiota, enhancing
its benecial prole. ese data also suggest that perhaps intestinal microbial “ngerprints” could be generated
to estimate vulnerability to metabolic dysfunction and/or the potential for ecacy of metabolic interventions.
Overall, data in the manuscript strongly suggest that the development of microbially-targeted therapies – both
primary and adjunctive – that are built upon safe, natural, plant-based products like fenugreek could be used to
attain signicant advancements in public health within the context of contemporary dietaryenvironments.
Materials and Methods
Animals and diets. is study was carried out in strict accordance with PHS/NIH guidelines on the use of
experimental animals, and all experimental protocols were approved by the Institutional Animal Care and Use
Committee at Pennington Biomedical Research Center. Data in this manuscript follows an initial publication on
the eects of whole fenugreek seed supplementation (2% w/w) on overall metabolic function in the context of a
16-week trial of high fat diet consumption41. As detailed in our initial report41, male, 9 week-old C57BL/6 J mice
were purchased from Jackson Laboratories, and group-housed (4/cage) under standard conditions with ad libi-
tum access to food/water. Aer 7 days acclimation, mice were randomly separated into the following 4 groups (20
mice each in each group): high fat diet ± fenugreek (HFD and HFD/FG) and control diet ± fenugreek (CD and
CD/FG) for 16 weeks. HFD and HFD/FG mice were fed a diet with 60% kcal from fat without or with 2% fenu-
greek seed powder incorporated into the diets, respectively (Research Diets Inc. D12492, D16020410), while CD
mice were fed a nutritionally matched control low fat diet (10% kcal from fat) with or without 2% fenugreek seed
powder (Research Diet Inc.; D12450J, D16020408). All diets contained 10% kcal from protein with the balance
in caloric intake provided by dierences in carbohydrate content. T. foenum-graecum L. “Fenugreek” seeds were
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purchased from Johnny’s Selected Seeds, Winslow Maine, certied organic for sprouting. Fenugreek seeds were
ground in the Department of Plant Biology at Rutgers University and hand-delivered to Research Diets Inc., (New
Brunswick, NJ) for commercial incorporation into treatment diets at 2% of the diet by weight.
Metabolic phenotyping. Assessment of metabolic function is fully detailed in our previous report41. Briey,
body composition (fat mass, fat-free/lean mass, and water content) was measured by briey placing in mice into
Bruker minispec LF110 time domain NMR analyzer (Bruker Optics, Billerica MA) as described previously41.
Glucose tolerance was measured using an oral glucose tolerance assay (OGTT) based on repeated sampling of tail
blood using a glucometer (Ascensia Elite, Bayer, Mishawaka, IN) at 0, 20, 40, 60, and 120 minutes aer oral glu-
cose (2 gm/kg) administration. All mice remained in the study for the duration of the 16-week feeding trail, aer
which mice with euthanized following a 8-hr fast by decapitation under deep isourane anesthesia. Levels of total
cholesterol, HDL cholesterol, LDL cholesterol, and triglycerides in serum collected at euthanasia were measured
colorimetrically (Wako Chemicals, Richmond, VA).
16S Metagenomic sequencing. Fecal samples were collected at euthanasia, and DNA preparation,
sequencing and bioinformatics were performed by the PBRC Genomics Core Facility. DNA was isolated
using a commercial reagent system (MoBio Power Fecal Kit, MoBio Laboratories, Carlsbad, CA) augmented
by enzymatic lysis using lysostaphin, mutanolysin, and lysozyme82. Sequencing libraries targeting V4 of the
gene encoding the 16S ribosomal RNA were generated using a commercially available kit (NEXTex 16S V4
Amplicon-Seq Library Prep Kit, BIOO Scientic, Austin, TX), relying on 16S gene-specic primer sequences V4F
5-GTGCCAGCMGCCGCGGTAA-3 and V4R 5-GGACTACHVGGGTWTCTAAT-3, and including Illumina
adaptors and molecular barcodes as described by the manufacturer to produce 253 bp amplicons. Samples were
sequenced with custom primers (BIOO Scientic, Austin, TX) on an Illumina MiSeq instrument using version
3 sequencing chemistry (300 bp paired end reads). Forward and reverse sequence reads were processed into
double-stranded DNA contigs using quality control metrics implemented in the soware package ‘mothur’83.
Sequence clustering (at better than 97% identity) to identify operational taxonomical units (OTUs), removal
of chimeric sequences, and generation of a read count table (i.e. tabulating the occurrence of each OTU in each
sample) were performed with the soware package ‘usearch84. Taxonomical classication of each OTU sequence
relied on the SILVA 16S rRNA sequence database version 123.185, and statistical tests for dierential representa-
tion were performed with tools incorporated in ‘mothur’, as well as using the soware package DESeq. 286.
Relative abundance of each OTU was examined on the phylum, class, order, family and genus levels.
Statistical analyses. Biochemical data were analyzed using Prism soware (GraphPad Soware, Inc.), and
displayed as mean ± standard error, and were analyzed by ANOVA. Statistical signicance for all analyses was
accepted at p < 0.05, and *, **, and *** represent p < 0.05, p < 0.01, and p < 0.001, respectively. Alpha diver-
sity (chao1 metrics) and beta diversity (weighted UniFrac metrics87) were assessed using tools implemented in
‘mothur’ on the basis of 80,000 sequences per sample. Dierential representation of OTUs was assessed using
DESeq. 2 on the basis of sequence count data, relying on Wald statistics with Benjamini-Hochberg correction
and a false discovery rate cuto set at 0.1. Inter-sample relationships relying on Principal Component Analysis
on the basis of DESeq. 2 output, and data visualizations were both performed using JMP Genomics soware
(SAS, Cary, NC). Pairwise Pearson correlations of individual metrics of metabolic function against individual
HFD-transformed, fenugreek-corrected OTU expression were carried out using Prism soware.
Received: 12 July 2019; Accepted: 7 January 2020;
Published: xx xx xxxx
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Acknowledgements
e authors gratefully acknowledge Dr. Kem Singletary and Ms. Cynthia Kloster for expert veterinary assistance
and insight into the described studies. ese studies were supported by grants from the National Center For
Complementary & Integrative Health and the Office of Dietary Supplements of the National Institutes of
Health (RO1AT010279 and P50AT002776; which funds the Botanical Dietary Supplements Research Center
of Pennington Biomedical Research Center and the Department of Plant Biology and Pathology in the School
of Environmental and Biological Sciences (SEBS) of Rutgers University). These studies also utilized the
facilities of the Animal Behavior/Phenotyping and Genomics Cores that are supported in part by COBRE (NIH
P20-GM103528) and NORC (NIH P30-DK072476) center grants from the National Institutes of Health.
Author contributions
A.B.K. and J.M.S. conceived the experiments. D.M.R. procured and performed all quality control on the fenugreek
seeds. S.F.K., A.J.R., and A.B.K. conducted the animal experiments. R.C. performed metagenomic sequencing,
while S.N., J.M.S., and A.B.K. analyzed the data. A.B.K. and J.M.S. prepared the manuscript and gures. All
authors reviewed the manuscript.
Competing interests
e authors declare no competing interests.
Additional information
Supplementary information is available for this paper at https://doi.org/10.1038/s41598-020-58005-7.
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