Pulse Crop Effects on Gut Microbial

Populations, Intestinal Function, and

Adiposity in a Mouse Model of
Diet-Induced Obesity John N. Mcginley
Visit to download the full and correct content document:
https://ebookmass.com/product/pulse-crop-effects-on-gut-microbial-populations-intest
inal-function-and-adiposity-in-a-mouse-model-of-diet-induced-obesity-john-n-mcginley
/
More products digital (pdf, epub, mobi) instant
download maybe you interests ...

New and Future Developments in Microbial Biotechnology

and Bioengineering: Crop Improvement through Microbial
Biotechnology 1st Edition Ram Prasad

https://ebookmass.com/product/new-and-future-developments-in-
microbial-biotechnology-and-bioengineering-crop-improvement-
through-microbial-biotechnology-1st-edition-ram-prasad/

A comparison process for mouse pairs John R. Steel

https://ebookmass.com/product/a-comparison-process-for-mouse-
pairs-john-r-steel/

Drug-Induced Ocular Side Effects: Clinical Ocular

Toxicology 8th Edition Frederick T. Fraunfelder

https://ebookmass.com/product/drug-induced-ocular-side-effects-
clinical-ocular-toxicology-8th-edition-frederick-t-fraunfelder/

Evaluation of the protein-sparing effects of

carbohydrates in the diet of the crayfish, Procambarus
clarkii Chuang Wen

https://ebookmass.com/product/evaluation-of-the-protein-sparing-
effects-of-carbohydrates-in-the-diet-of-the-crayfish-procambarus-
clarkii-chuang-wen/
In the Shadow of Humanity: A Novel N. John Williams

https://ebookmass.com/product/in-the-shadow-of-humanity-a-novel-
n-john-williams/

Lymphatic Structure and Function in Health and Disease

1st Edition Felicity N. E. Gavins (Editor)

https://ebookmass.com/product/lymphatic-structure-and-function-
in-health-and-disease-1st-edition-felicity-n-e-gavins-editor/

Gum Arabic-enriched diet modulates growth, antioxidant

defenses, innate immune response, intestinal microbiota
and immune related genes expression in tilapia fish
Mohammed A.E. Naiel
https://ebookmass.com/product/gum-arabic-enriched-diet-modulates-
growth-antioxidant-defenses-innate-immune-response-intestinal-
microbiota-and-immune-related-genes-expression-in-tilapia-fish-
mohammed-a-e-naiel/

A Unified Treatment of Moore's Paradox John N. Williams

https://ebookmass.com/product/a-unified-treatment-of-moores-
paradox-john-n-williams/

Obesity and Gynecology 2nd Edition Tahir A. Mahmood

(Editor)

https://ebookmass.com/product/obesity-and-gynecology-2nd-edition-
tahir-a-mahmood-editor/
Article

Pulse Crop Effects on Gut Microbial Populations,

Intestinal Function, and Adiposity in a Mouse Model
of Diet-Induced Obesity
John N. McGinley 1, Vanessa K. Fitzgerald 1, Elizabeth S. Neil 1, Heather M. Omerigic 2,
Adam L. Heuberger 2, Tiffany L. Weir 3, Rebecca McGee 4,
George Vandemark 4 and Henry J. Thompson 1,*
1 Cancer Prevention Laboratory, Colorado State University, Fort Collins, CO 80523, USA;
john.mcginley@colostate.edu (J.N.M.); vanessa.fitzgerald@colostate.edu (V.K.F.);
elizabeth.neil@colostate.edu (E.S.N.)
2 Department of Horticulture, Colorado State University, Fort Collins, CO 80523, USA;

heather.omerigic@colostate.edu (H.M.O.); adam.heuberger@colostate.edu (A.L.H.)

3 Department of Food Science and Human Nutrition, Colorado State University,

Fort Collins, CO 80523, USA; tiffany.weir@colostate.edu (T.L.W.)

4 USDA-ARS Grain Legume Genetics and Physiology, Washington State University,

Pullman, WA 99164, USA; rjmcgee@wsu.edu (R.M.); george.vandemark@ars.usda.gov (G.V.)

* Correspondence: henry.thompson@colostate.edu; Tel.: +1-970-491-7748; Fax: +1-970-491-3542

Received: 28 December 2019; Accepted: 21 February 2020; Published: 25 February 2020

Abstract: The dietary fiber gap that is present in many countries co-exists with a low intake of grain
legumes (pulses) that have 2–3 times more dietary fiber than cereal grains that are commonly
recommended to increase fiber intake. Given the relationships among dietary fiber, gut health and
chronic disease risk, a study was undertaken in a preclinical mouse model for obesity to examine
how commonly consumed pulses, i.e., chickpea, common bean, dry pea and lentil, would impact
gut microbes, intestinal function, and adiposity. Pulses were fed to C57BL/6 mice at similar levels
of protein and fiber. Bacterial count in the cecum was elevated 3-fold by pulse consumption. At the
phylum level, a 2.2- to 5-fold increase in Bacteriodetes relative to Firmicutes was observed. For
Akkermansia muciniphila, a health-beneficial bacterium, differential effects were detected among
pulses ranging from no effect to a 49-fold increase. Significant differences among pulses in
biomarkers of intestinal function were not observed. Pulses reduced accumulation of lipid in
adipose tissue with a greater reduction in the subcutaneous versus visceral depots. Metabolomics
analysis indicated that 108 metabolites were highly different among pulse types, and several
compounds are hypothesized to influence the microbiome. These results support recent
recommendations to increase consumption of pulse-based foods for improved health, although all
pulses were not equal in their effects.

Keywords: adiposity; Akkermansia muciniphila; Bacteriodetes; chickpea; common bean; dry pea;
Firmicutes; intestinal function; lentil; pulses

1. Introduction
In many regions of the world, grain legumes, also referred to as pulses, are neglected staple
foods that have the potential to directly impact food insecurity while helping individuals achieve
healthy and balanced diets and to accelerate progress towards the Sustainable Development Goals of
the WHO/FAO [1–5]. While pulses are frequently promoted for their content of protein and
micronutrients, they are also an excellent source of dietary fiber, a fact that is under appreciated [6].
Nutrients 2020, 12, 593; doi:10.3390/nu12030593 www.mdpi.com/journal/nutrients
Nutrients 2020, 12, 593 2 of 17

Since the seminal work of Burkitt and colleagues, there has been an awareness of the role of dietary
fiber in maintaining the physiological function of the mammalian intestine [7]. However, recognition
of the importance of dietary fiber for human health and well-being has never risen to the
establishment of fiber as an essential nutrient. Nonetheless, a recommended level of dietary fiber has
been proposed (14 g/1000 kcal) and many systematic reviews and meta-analyses support the health
benefits associated with cohorts that meet or exceed this recommendation [8–13]. Unfortunately, the
majority of individuals in developed countries such as the United States fail to meet the
recommended level of intake for dietary fiber and the magnitude of the gap is large, an approximately
50–70% shortfall [6]. This gap has been resistant to change, despite decades of public health
interventions and development of fiber enriched food products. Recently, we have advocated more
focus on pulse crops to not only close the dietary fiber gap, but to extend intake above recommended
levels to those that have been reported to significantly reduced chronic disease mortality [14].
However, to achieve such intakes, i.e., 50–80 g total dietary fiber per day, we argue that a better
understanding of the effects of high fiber foods such as pulses is needed. We recently published a
comparative analysis of the dietary fiber content of chickpea, common bean, dry pea, and lentil, and
reported that differences existed among pulses that may be of importance to various sectors of the
food systems industry [6,15]; however, from the consumers’ perspective, none of the differences
observed negate the conclusion that pulses are an abundant source of dietary fiber.
Of the many functions that dietary fiber may affect in mammalian species, there is considerable
interest in its influence on the meta-organism, i.e., the gut-associated microbiome [16–20]. The
available literature on this topic is expanding rapidly, e.g., [21–24]; however, we found no studies in
which the effects of the four most predominant pulses have been compared. This is important for
several reasons. First, there is no consensus on what component(s) of dietary fiber are essential for
gut health (reviewed in [6]), and consequently, various reports of dietary fiber composition [21,25–
28] cannot be used to predict gut health effects. Second, a considerable body of literature indicates
that gut health effects are attributable to microorganisms that are obligate anaerobes. Thus, except
for one report in which microbial analyses were performed on cecal content and stool [21], analyses
of the microbiome have been limited to fecal material which may not be directly representative of the
profile of microorganisms that colonize the intestinal tract of pulse fed animals/humans and that are
responsible for effects of the microbiome on health status. Third, it is widely recognized that results
of microbiome analyses can vary widely among publications for a host of technical reasons unrelated
to the underlying biology and biochemistry being investigated [29–31]; this makes direct comparisons
among pulses within the same study of considerable value. Finally, while investigation of cultivars
of various pulses is very important, it is equally important to also move the field in a translational
direction, i.e., the analysis of each pulse crop as a collection of cultivars that are agronomically
important and as might be eaten by the consumer, an approach that agricultural economists refer to
as a market basket. In view of these gaps in research, the study reported herein was designed to
address three questions when pulses were fed at a similar level of dietary protein and fiber. Do the
four pulses: (1) exert the same effect on specific microbial populations that have been implicated in
health promotion/disease prevention, (2) impact intestinal function in the same manner, and (3) affect
adipose tissue deposition in a mouse model of diet-induced obesity?

2. Materials and Methods

2.1. Experimental Animals

NCI C57BL/6NCr male mice (21–28 days of age) were obtained from Charles River Laboratories
NCI (Frederick, MD, USA). Upon arrival, the mice were fed a purified high-fat diet. This formulation
was developed and first reported to induce obesity in this mouse strain by Ulman et al., an effect
reported to be accompanied by dysbiosis [32–37]. The formulation, which provides 60% of dietary
kcal from fat, is commercially available (Catalogue #D12492, Research Diets, Inc., New Brunswick,
NJ, USA) [38]. Mice were housed in solid-bottomed polycarbonate rodent cages (42.55 cm long × 20.32
cm wide × 20.32 cm high) that accommodate up to 10 adult mice per cage as defined in the Guide for
Nutrients 2020, 12, 593 3 of 17

Use of Laboratory Animals [39]. The animal bedding used was 7090 Teklad sani-chips from Envigo
(Indianapolis, IN, USA ). Mice were maintained on a 12 h light/dark cycle at 27.5 ± 2 °C with 30%
relative humidity and given ad libitum access to diet and distilled water. All animal studies were
performed in accordance with the Colorado State University Institutional Animal Care and Use
Committee (protocol 18-7746A).

2.2. Experimental Design

As described above, mice were fed the Ulman diet from receipt to 5 weeks of age in order to
promote dysbiosis that is associated with the obesity phenotype which manifests beginning 8 weeks
of age [40]. At 5 weeks of age, mice were randomized to treatment groups. Mice were either continued
on the high-fat (HF) formulation (control diet) or were fed that diet formulation to which common
bean, chickpea, dry pea, or lentil was added. The diet formulation has been previously reported [41]
and therefore the formulation is detailed in Supplementary Table S1. The formulation of the
experimental diets and the rationale for the concentration of pulses has been published [42,43].
Briefly, the level of pulse was (40% w/w), which is approximately twice as high as median intakes of
pulse consuming subgroups in the US and Canada. However, the dietary level is actually similar to
dietary amounts consumed in developing countries where pulses are dietary staples [43]. Key aspects
of the dietary approach were: (1) the inclusion of a low-fat, negative control group, which varied
markedly in composition from the high-fat control but that is a recognized control in the B6 DIO
model in that it does not induce dysbiosis and has lower obesogenic activity [44], (2) the use of a
single common bean market class as a “gold standard” against which to compare the activity of other
pulses since we have reported that this common bean market class has anti-cancer and anti-
obesogenic activity and that it induces favorable changes in gut microbial populations in this model
system [41,45,46], and (3) the creation and investigation of chickpea, dry pea, and lentil using a
market basket approach of cultivars that are economically important, but whose identity is unknown
by the consumer; thus this reflects what the consumer is likely to experience. Diets were formulated
to be similar in protein using proximate analysis data available to the consumer (Supplementary
Table S2). Diets containing pulses were supplemented with both the sulfur-containing amino acid,
methionine, and with tryptophan, which are respectively the first and second most limiting amino
acids in pulse crops [47].
Mice were rehoused at randomization in a single cage per group. Because of aggressive behavior
among a few mice, aggressors were removed to prevent injury to the other mice thus resulting in 6–
8 mice per group. Mice were fed their respective diets ad libitum throughout the experiment. Body
weight data were collected weekly. The experiment was terminated at 22 weeks of age when animals
had consumed experimental diets for 17 weeks.

2.3. Necropsy
All the animals in each group were subjected to the following procedures. Mice were not fasted
prior to necropsy. Mice were anesthetized using isoflurane inhalation and subsequently euthanized
by cervical dislocation. The gastrointestinal tract including the stomach to the superior portion of the
rectum was removed. The small intestine was cut at the ileocecal junction and weighed with the
stomach attached. A 6.5 cm length of ileum superior to the cecum was excised. A 1 cm section of
flushed ileum immediately superior to the cecum was cut and placed in 10% neutral buffered
formalin. The remaining ileum was laid on a Kimwipe and a midline incision was made down the
entire long axis using blunt tipped scissors. The ileum was reflected, and the edges of the serosa
gently tacked down to the Kimwipe. While holding one end of the ileum securely with Adson forceps,
a polished stainless-steel spatula was used to gently scrape the ileum mucosa in one continuous
motion; contents were placed into a cryovial and snap-frozen in liquid nitrogen for subsequent RNA
analysis. The cecum was cut from the colon, weighed and snap-frozen in liquid nitrogen without
removal of content in order to retain anaerobiosis with the cecal content. The colon was rinsed in
sterile saline and fecal pellets extruded while maintaining proper anatomic orientation. 1 cm sections
of colon representing the ascending, transverse and descending portions was excised and fixed in
Nutrients 2020, 12, 593 4 of 17

10% neutral buffered formalin. Inguinal subcutaneous and abdominal visceral adipose tissue were
harvested and weighed. In addition, a small portion of these adipose depots were excised and fixed
in 10% neutral buffered formalin. Remaining portions of fat depots were snap-frozen in liquid
nitrogen. All snap-frozen tissues were stored at −70 °C. The left tibia of each animal was removed,
cleaned and measured in mm using a pair of digital calipers in order to normalize the tissue weights.
All formalin fixed tissues were processed for histological evaluation.

2.4. RNA Transcript Expression

RNA was isolated from frozen mucosal scrape of the ileum using the RNeasy Mini kit (Qiagen,
Germantown, MD, USA) according to the manufacturer’s protocol. Isolated RNA purity (260/280 and
260/230 ratios) and concentration was checked via NanoDrop (Thermo Fisher Scientific, Waltham,
MA, USA). RNA integrity was determined using a Bio-Rad Experion (Bio-Rad, Hercules, CA, USA).
cDNA was synthesized from 1 µL of total RNA using Superscript II reverse transcriptase (Invitrogen,
Carlsbad, CA, USA). DNA oligo primers were synthesized (Integrated DNA Technologies, Coralville,
IA, USA), sequences are shown in Supplementary Table S3 [48]. Quantitative real-time PCR was
performed on an iCycler (Bio-Rad, Hercules, CA, USA) with optical-grade 96-well plates (Thermo
Fisher Scientific, Waltham, MA, USA). Each 20 µL reaction mixture was composed of 5 µL of water,
10 µL 2X SYBR green Supermix (Bio-Rad, Hercules, CA, USA), 1.5 µL each of forward and reverse
primers at a final concentration of 0.3 µM, and 2 µL of synthesized cDNA. The following PCR
conditions were used: 95 °C for 3 min, followed by 40 cycles of 95 °C for 15 s and 56.5 °C for 1 min.
Fluorescent products were detected at the last step of each cycle. qPCR was followed by a melting
curve analysis to verify primer specificity under the following conditions: 80 cycles starting at 57 °C
for 10 s and increasing by 0.5 °C each cycle. Each sample was run in triplicate and the relative
farnesoid X receptor (FXR) expression was calculated by normalizing the threshold cycle (Ct) values
to 18S [48]. The fold change in expression relative to the control-fed animals was computed by the
delta, delta Ct method as described in [49].

2.5. Bacterial Quantification by qPCR

An established approach was used to assess levels of specific bacterial populations. DNA oligo
primers coding for 16sRNA sequences unique to Akkermansia muciniphila, and bacteria in the phyla
Firmicutes and Bacteroidetes, or common to all bacteria were used for qPCR analysis. The DNA
oligomers were synthesized by Integrated DNA Technologies, Coralville, IA, and are shown in
Supplementary Table S3 [50–52].
Frozen ceca were removed from the freezer three at a time and allowed to slightly thaw on the
bench top, approximately 5–10 min. Small scissors were used to make an incision in the cecum and
the mucosa was reflected. A clean, polished stainless-steel spatula was used to remove approximately
100 mg of cecal contents and placed in a dry bead tube and DNA extracted according to the
manufacturer’s protocol, QIAamp PowerFecal DNA kit (Qiagen, Germantown, MD, USA). The
isolated DNA was checked for purity (260/280 and 260/230 ratios) and concentration via NanoDrop
(Thermo Fisher Scientific, Waltham, MA, USA).
qPCR was performed on cecal DNA using a modified version of the protocol described in [51].
Briefly, qPCR amplification and detection were performed using an iCycler (Bio-Rad, Hercules, CA,
USA) with optical-grade 96-well plates (Thermo Fisher Scientific, Waltham, MA, USA). Each 20 µL
reaction mixture was composed of 5 µL of water, 10 µL 2X SYBR green Supermix (Bio-Rad, Hercules,
CA, USA), 1.5 µL of forward and reverse primers at a final concentration of 0.3 µM, and 2 µL of
template DNA (10 ng/µL). PCR conditions were as follows: 95 °C for 3 min, followed by 40 cycles of
95 °C for 15 s, 60 °C for 30 s, and 72 °C for 30 s, and a final extension at 72 °C for 5 min. Fluorescent
products were detected at the last step of each cycle. qPCR was followed by a melting curve analysis
to verify primer specificity under the following conditions: 80 cycles starting at 5 5 °C for 10 s and
increasing by 0.5 °C each cycle. Each sample was run in triplicate, Ct values were normalized to 16S
(926 Fwd., 1062 Rev.), and the fold change in expression relative to the control was computed by the
delta, delta Ct method as described in [49].
Nutrients 2020, 12, 593 5 of 17

2.6. Histology

2.6.1. Histology and Image Acquisition

The tissue fixed in 10% neutral buffered formalin for 24 hours was then processed through a
series of graded ethanols, cleared in toluene, embedded in paraffin and sections were cut at 4 µM.
Sections were stained with H&E, alcian blue nuclear fast red (AB) [53], and Ki-67 immunostaining as
described previously [54]. Images of stained intestine were captured at 200× and images of adipose
tissue at 100x magnification using a Zeiss AxioCamHR (Zeiss, Thornwood, NY, USA) digital camera
mounted on a Zeiss Axioskop II microscope. Prior to capturing images, background shading
correction was achieved using the Zeiss AxioVision software. Images were captured (1300 × 1030
pixels, 24 bit RGB, 150 DPI) and saved as JPEG files.

2.6.2. Morphometric Analysis

A total of ten crypts in ileum, and ascending, transverse and descending colon were analyzed
from each animal across serial sections of three different stains listed in section 2.7.1. Crypts were
chosen from multiple image fields along the length of each segment in order to decrease bias and
selection was based on the following criteria: (1) a single layer of epithelial cells visible from the base
of the crypt to the top of the of villus; (2) patent crypt lumen; (3) visible crypt lamina propria. Ileum
and colon crypt height was measured manually from the base of the crypt to the brush border of the
uppermost enterocyte nearest the lumen in H&E-stained images using the caliper function in Image
Pro Plus v4.5 (Media Cybernetics, Inc. Rockville, MD). AB-stained images depicting crypt goblet cells
and intra-crypt mucin were processed using the built-in alcian blue vector in the color deconvolution
plugin for ImageJ v1.52d (NIH, Bethesda, MD), resulting in three separate color channel images, red,
green and blue. A macro was written in Image Pro Plus v4.5 (Media Cybernetics, Inc. Rockville, MD)
to analyze the AB images. Briefly, the blue channel image was imported along with the original AB
image. The blue channel image was converted to 8 bit grayscale (256 shades of gray) and contrast
applied resulting in a black and white image. Crypts were circumscribed one at a time on the original
AB image as an area of interest (AOI). The AOI was copied from the original AB image, pasted to the
corresponding XY coordinates in the black and white image and the segmentation range was set 0–
254, i.e. black representing alcian blue stained mucin within the crypt against a white (255)
background. The software measured the total sum black area in µM2 of the AOI and exported the
value automatically via dynamic data exchange to an Excel spreadsheet for analysis. To assess cell
proliferation, Ki-67 staining was performed as previously described [54] and analyzed using the
immunoratio plugin for ImageJ [55].

2.6.3. Metabolite Extraction, Detection, and Data Processing

A total of 100 mg of cooked pulse powder was extracted using a MTBE/methanol/water
procedure and detected on liquid chromatography–mass spectrometry (LC–MS, positive and
negative electrospray ionization modes) and gas chromatography–mass spectrometry (GC–MS,
trimethylsilyl/methoximation derivatizations, liquid injections) as described in [56,57]. The data
processing procedure is described in [11,58,59]. Briefly, for each sample, raw data files were
converted to .cdf format, and a matrix of molecular features as defined by retention time and mass
(m/z) was generated using XCMS software in R for feature detection and alignment. Features were
grouped using RAMClustR with TIC normalization. LC–MS data were first annotated by searching
against an in-house spectra and retention time database using RAMSearch. RAMClustR was used to
call the findMain function from the interpretMSSpectrum function (MS-FINDER program v2.40
[60,61]) to infer the molecular weight of each LC–MS compound for annotation of the mass signals.
The complete MS spectrum and a truncated MSE spectrum were written to a .mat format for import
to MSFinder. The MSE spectrum was truncated to include only masses with values less than the
inferred M plus its isotopes, and the .mat file precursor ion was set to the M+H ion for the findMain
inferred M value. These .mat spectra were analyzed to determine the most probable molecular
Nutrients 2020, 12, 593 6 of 17

formula and structure. MSFinder was also used to perform a spectral search against the FooDB,
HMDB, ChEBI, and Lipidmaps metabolite databases (foodb.ca; hmdb.ca; ebi.ac.uk/chebi;
lipidmaps.org). All results were imported into R and a collective annotation was derived with
prioritization of MSFinder mssearch > MSFinder structure > MSFinder formula > findMain M. All R
work was performed using R version 3.3.1 [6]. GC–MS data were annotated using spectral and
retention index matching within AMDIS software using the Golm Metabolome Database [62].

2.7. Statistical Analyses

Body mass and morphometric data were evaluated by ANOVA or the Kruskal–Wallis test
depending on data distribution (D’Agostino and Pearson omnibus normality test) and Fisher or
Dunn’s post hoc test, respectively. Differences were considered significant at p ≤ 0.05. Bacterial, and
RNA transcript expression data were evaluated by computing fold change (FC) and the significant
of differences tested after log transformation and boot strapping to generate confidence intervals [63].
Data analyses were conducted using Systat version 13.0, SAS version 9.2, and Graph Pad Prism,
version 5.2. Principal component analysis and orthogonal projection to latent structures analysis of
metabolites were conducted using SIMCA v15 software (Sartorius Stedim Biotech, Umea, Sweden),
on unit variance scaled data. z for each metabolite was calculated as the mean metabolite abundance
value within each pulse type minus the mean abundance values of all pulse types divided by the
standard deviation of the abundance value all pulse types.

3. Results

3.1. Effects of Pulses on Growth

Mice were assigned to diet groups via the staggered randomization approach using body
weight. Thus, group mean body weights were essentially identical when the feeding of experimental
diets was initiated at 5 weeks of age. Mice were weighed weekly as shown in Figure 1. At no time
point during the study were differences found to be statistically significant among treatment groups.

Figure 1. Effect of feeding pulse diets on body weight. HF Control n = 7, LF Control n = 7, Bean n = 6,
Chickpea n = 7, Dry Pea n = 8, Lentil n = 8; HF: high fat; LF: low fat. Values are means ± SEM.
Differences among groups at each timepoint were not statistically significant.

3.2. Effect of Pulses on Cecal Bacteria Populations

Pulse consumption induced a 3-fold increase in the bacterial content of the cecum relative to the
content measured in the high-fat control group (Figure 2); no differences were noted among the
pulses evaluated. The amount of Akkermansia muciniphila in the cecum relative to the high-fat control
group was markedly elevated in bean- and lentil-fed mice but was unaffected in chickpea or dry pea-
fed mice. Selective effects were also noted on bacteria in the phylum Firmicutes (no effect to
Nutrients 2020, 12, 593 7 of 17

suppression) and Bacteroidetes (increased 2.3- to 3.0-fold). When the fold change data were expressed
as a ratio, the increase in Bacteroidetes to Firmicutes was increased 2.3 to 5.0 in pulse consuming
mice, with the greatest increase in lentil-fed mice.

Figure 2. Effect of feeding pulse diets on cecal bacteria populations. (A) 16S Ct for 20 ng genomic
DNA, mean ± SEM; ANOVA:LF Control p < 0.0001, Bean p < 0.0001, Chickpea p < 0.0001, Dry Pea p <
0.0001, Lentil p < 0.0001; HF Control vs. all pulses p < 0.0001; LF Control vs. all pulses p = 0.0443 (B)
Akkermansia muciniphilia ∆Ct, ANOVA: LF Control p = 0.0071, Bean p = 0.0001, Chickpea p = 0.9300,
Dry Pea p = 0.2131, Lentil p < 0.0001; HF Control vs. all pulses p = 0.1342; LF Control vs. all pulses p =
0.0006 (C) Firmicutes ∆Ct, ANOVA: LF Control p = 0.7762, Bean p = 0.0370, Chickpea p = 0.1450, Dry
Pea p = 0.8696, Lentil p < 0.0052; HF Control vs. all pulses p = 0.0937; LF Control vs. all pulses p = 0.1497
(D) Bacteroidetes ∆Ct, ANOVA: LF Control p = 0.0003, Bean p < 0.0001, Chickpea p = 0.0001, Dry Pea
p < 0.0001, Lentil p < 0.0001; HF Control vs. all pulses p = < 0.0001; LF Control vs. all pulses p = 0.2189;
FC relative to HF control diet; *different relative to HF control diet (p ≤ 0.05); #different relative to LF
control diet (p ≤ 0.05); FC calculated relative to HF Control; HF Control n = 7, LF Control n = 7, Bean n
= 6, Chickpea n = 7, Dry Pea n = 8, Lentil n = 8; FC: fold change; HF: high fat, LF: low fat.

3.3. Effect of Pulses on Ileal FXR Expression

Total RNA was isolated from the ileum and the level of FXR transcripts was determined by
qPCR relative to 18S expression. Overall, pulse consumption increased FXR transcript levels relative
to the high-fat control group by approximately 2-fold (p < 0.05); however, the differences among
pulses were not statistically significant (Figure 3).

Figure 3. Farnesoid X Receptor (FXR) expression. (A) 18S Ct, ANOVA: LF Control p = 0.0268, Bean p
= 0.9537, Chickpea p = 0.3215, Dry Pea p = 0.5767, Lentil p = 0.2114; (B) FXR ∆Ct, ANOVA: LF Control
Nutrients 2020, 12, 593 8 of 17

p = 0.6239, Bean p = 0.1225, Chickpea p = 0.2717, Dry Pea p = 0.1044, Lentil p = 0.0787; * different relative
to HF Control (p ≤ 0.05); # different relative to LF Control (p ≤ 0.05). Fold change was calculated relative
to HF Control; ANOVA: HF Control vs. all pulses p = 0.05, LF Control vs. all pulses p = 0.01, HF
Control vs. LF Control p = 0.26; HF Control n = 7, LF Control n = 7, Bean n = 6, Chickpea n = 7, Dry Pea
n = 8, Lentil n = 8; HF: high fat; LF: low fat.

3.4. Effect of Pulses on Intestinal Morphometry

In order to further assess the effects of pulse consumption on intestinal function, morphometric,
histochemical, and immunohistochemical techniques were applied to the ileum, and the ascending,
transverse, and descending segments of the colon. The nature of these measurements as applied to
the indicated intestinal segments is illustrated in Supplementary Figure S1. Only crypt height was
affected by pulse consumption (Table 1). Pulses induced some changes in alcian blue staining and in
the labeling index, but since these effects did not reach the level of statistical significance, the results
are provided in Supplementary Table S4 and Table S5, respectively. Extension of crypt height was
observed in pulse-fed mice in the transverse and descending segments of the colon. The magnitude
of extension varied by pulse with the greatest extension being noted in lentil-fed mice. It is
noteworthy that the longer crypts were not associated with either a change in mucin content of goblet
cells (Supplementary Table S4) or a change in the magnitude of cell proliferation (Supplementary
Table S5).

Table 1. Effect of feeding pulses on intestinal crypt height.

Diet 1 Ileum Ascending Colon Transverse Colon Descending Colon

High-fat control 252.1 ± 41.1 a 92.2 ± 9.3 b 182.2 ± 23.7 f 135.9 ± 9.6 k
Low-fat control 262.4 ± 18.0 a 93.3 ± 6.9 b, c 207.3 ± 34.1 f, g 150.1 ± 10.4 k, l
Bean 274.5 ± 29.5 a 82.2 ± 11.7 d 214.1 ± 13.1 f, g, h 153.9 ± 16.6 l, m
Chickpea 269.7 ± 35.4 a 89.5 ± 6.3 b, c, d 239.1 ± 50.6 g, i 139.6 ± 17.1 k, l, m
Dry Pea 272.2 ± 27.5 a 77.7 ± 7.6 d, e 244.2 ± 53.2 g, i 152.7 ± 14.6 l, m
Lentil 255.6 ± 24.5 a 86.9 ± 8.4 b, c, d 257.6 ± 23.6 i, j 166.0 ± 19.1 m, n
p-values
0.4676 0.0182 0.0015 0.0525
1Values are crypt height (µm2) means ± SD; data compared using ANOVA and the Fisher least
significant difference method for pairwise comparisons; different letter superscripts within a column
indicate statistical significance p ≤ 0.5; Ileum ANOVA: HF Control vs. all pulses p = 0.22, LF Control
vs. all pulses p = 0.69, HF Control vs. LF control p = 0.52; Ascending colon ANOVA: HF Control vs. all
pulses p = 0.04, LF Control vs. all pulses p = 0.02, HF Control vs. LF control p = 0.82; Transverse colon
ANOVA: HF Control vs. all pulses p < 0.01, LF Control vs. all pulses p = 0.04, HF Control vs. LF control
p = 0.22; Descending colon ANOVA: HF Control vs. all pulses p = 0.02, LF Control vs. all pulses p =
0.63, HF Control vs. LF Control p = 0.12; Pulses include bean, chickpea, dry pea and lentil; High-fat
control n = 7, Low-fat control n = 7, Bean n = 6, Chickpea n = 7, Dry Pea n = 8, Lentil n = 8; HF: high fat,
LF: low fat.

3.5. Effect of Pulses on Adiposity

Pulse consumption decreased both subcutaneous and visceral fat mass relative to the high-fat
control, but fat mass was greater than that of the low-fat control group. Differences among pulses
were not statistically significant. The reduction in fat mass was greater in the subcutaneous versus
the visceral fat depot (Table 2).

Table 2. Effect of pulses on adiposity.

Subcutaneous Fat Sum visceral Fat Tibia

Diet 1 (mg/mm) 2 (mg/mm) 2 (mm)
High-fat control 138.7 ± 22.1 a 246.5 ± 25.8 d 17.8 ± 0.2 g, h, k, l
Low-fat control 74.3 ± 13.8 b 169.4 ± 14.7 e 17.9 ± 0.2 h, k
Nutrients 2020, 12, 593 9 of 17

Bean 122.9 ± 15.8 c 231.8 ± 8.2 d, f 17.3 ± 0.5 i, j, k, l

Chickpea 124.1 ± 22.1 c 229.2 ± 22.6 f 17.1 ± 0.3 j
Dry Pea 119.2 ± 21.2 c 226.6 ± 15.7 f 17.6 ± 0.3 k, l
Lentil 123.4 ± 20.8 c 229.7 ± 22.2 d, f 17.5 ± 0.3 l
p-values
< 0.0001 < 0.0001 0.0009
1 2
Values are means ± SD; data compared using ANOVA with tibia length as a covariate and the Fisher
least significant difference method for pairwise comparisons; different superscripts within a column
indicate statistical significance p ≤ 0.5; ANOVA for subcutaneous fat: HF Control vs. all pulses p =
0.05, LF Control vs. all pulses p < 0.01, HF Control vs. LF Control p < 0.01; ANOVA for sum visceral
fat: HF Control vs. all pulses p = 0.03, LF Control vs. all pulses p < 0.01, HF Control vs. LF Control p <
0.01; Pairwise comparisons: values within a column with different superscripts were statistically
significant, p < 0.05. Pulses include bean, chickpea, dry pea and lentil; High-fat control n = 7, Low-fat
control n = 7, Bean n = 6, Chickpea n = 7, Dry Pea n = 8, Lentil n = 8.

3.6. Metabolite Variation among Pulse Types

Metabolite variation among pulse types was evaluated using a non-targeted mass spectrometry
metabolomics approach. A total of 2433 metabolites were annotated and analyzed using principal
component analysis (PCA). PCA characterized the lentil cultivar Pardina as an outlier and it was
removed for all subsequent analysis. PCA with the outlier removed resulted in six components that
explained 71% of the variation. The PCA demonstrated most of the variation as attributed to
metabolite differences between pulse types (Figure 4). Specifically, PC1, PC2, and PC5 showed
separation of the four pulse types, although bean and chickpea were highly similar. The PC5
separation of bean and chickpea (4.1%) was due to a lack of compounds in chickpea, and only one
compound was identified to be higher in chickpea compared to the other pulses. Orthogonal
projection to latent structures discriminant analysis (OPLS-DA) was performed to identify
metabolites that were distinct to each pulse type, with bean, chickpea, dry pea, and lentil as the four
classes within the model. The OPLS-DA resulted in three components that explained R2Y = 99% of
the variation (Q2 = 89%). Analysis of the OPLS-DA loadings resulted in the characterization of 108
metabolites that were associated with differentiating the four pulse types (Supplementary Table S6
and Table S7).

Figure 4. Metabolite variation among pulse types. Principal component analysis (PCA, top) and
orthogonal projection to latent structures discriminant analysis (OPLS-DA, bottom) was performed
on 17 varieties spanning four pulse types. The data matrix consisted of 2433 metabolites for n = 2
replicates per variety. Figures are scores plots for select components of the PCA and OPLS-DA.
Nutrients 2020, 12, 593 10 of 17

4. Discussion
Foods are considered dietary staples when they are eaten in large amounts throughout the day
as a primary source of calories and protein. These foods are also affordable and accessible to the
populations in which they serve as dietary staples. Grain legumes, also referred to as pulses, have
been dietary staples since the dawn of agriculture [15]. However, in recent years a growing trend has
been to replace them with animal derived foods and protein isolates [64]. Consequently, in many
developed countries, pulses are now consumed infrequently by most of the population [2,65]. There
are four pulse crops that are predominant in the world’s food supply: common bean, chickpea, dry
pea, and lentil [15,66]. While most attention is given to pulses because they supply a large amount of
protein in the absence of lipid, they are also an exceptional source of dietary fiber, containing 2–3
times more total fiber per 100 kcal edible portion than other foods commonly promoted as rich
sources of dietary fiber [6,25,67].
Many barriers have obstructed progress in understanding how dietary fiber impacts human
health. Prominent among these issues has been the lack of an internationally accepted consensus
definition of dietary fiber and of an analytical approach that measures what is contained in that
definition [68]. Consequently, the publication of a consensus definition and method in 2009 and 2011,
respectively, was a landmark development in the field [69–71]. The definition and method divide
dietary fiber components into three major categories: insoluble dietary fiber, soluble dietary fiber,
and oligosaccharides. The variation among foods in these components as well as the investigation of
human consumption in terms of these dietary fiber categories has been used to reflect differences in
dietary fiber quality, but their association with health outcomes is controversial, and most
recommendations continue to be framed in terms of total dietary fiber intake [6]. Nonetheless, given
our recent work indicating that common bean, chickpea, dry pea, and lentil have similar levels of
total dietary fiber [25], we decided that a comparative investigation of these pulse would provide an
opportunity to compare the effects of similar amounts of dietary fiber from botanically similar but
distinct sources. As discussed in the following sections, what is unknown is whether the bioactivity
of pulses is equivalent.
A number of recent studies have reported the effects of the consumption of various pulse crops
on the fecal microbiome in preclinical models and numerous beneficial changes have been reported,
although comparative studies among pulses have not been undertaken [19,22,24,72]. A major
limitation of efforts to study the gut-associated microbiome in fecal specimens is that the majority of
the commensal microbial species that have important effects are obligate anaerobes and the stability
of these populations in feces is variable and appears to be short [73]. Recognizing this situation, and
the report of differences between the cecal and fecal microbiome in mice fed chickpea [21], the
bacterial analyses reported herein were made on the content of the cecum that was excised intact and
immediately frozen in liquid nitrogen to preserve the anaerobic status of its content. The cecum is a
segment of the gut that supports a diverse population of commensal microorganisms. Because the
cecum sits at the intersection of the small and large intestine, its microbial content reflects the impact
of dietary constituents that escape digestion in the small intestine. Consistent with expectations of a
high fiber diet, rich in insoluble and soluble fermentable carbohydrates, the overall content of bacteria
in the cecum was similarly increased by all pulses evaluated. Differential effects were also observed
for the specific bacterium Akkermansia munciniphila. While lentil and common bean induced,
respectively, a 49-fold and 25-fold increase in this bacterium relative to the high-fat control diet,
chickpea and dry pea had no effect. The relevance of A. muciniphila is that colonization of the gut with
this bacterium has been reported to be inversely associated with obesity, diabetes, and inflammation
[74–79]. The differential effects of pulse consumption on the content of A. muciniphila is consistent
with a selective effect limited to lentil and common bean. Given the emerging evidence of A.
muciniphila’s health benefits, the identification of the beneficial prebiotic components of pulse crops
is essential for at least two reasons. First, for lentil and common bean, in which the effect on A.
muciniphila ranged between a 25- and 49-fold increase (Figure 2B), knowledge of prebiotic
components could not only lead to the identification of cultivars within each crop with even stronger
colony promoting activity, but could also prevent the loss of those compounds during food
Nutrients 2020, 12, 593 11 of 17

processing and the development of new food products. We also observed that pulses had differential
effects on relative levels of bacteria in the phyla Bacteriodetes (increased) and Firmicutes (decreased).
Given the controversial nature of the literature indicating whether an increase in the Bacteroidetes to
Firmicutes ratio is consistent with health benefits [52,80–84], the importance of this observed is
unclear. Nonetheless, the rank order of increase in comparison to the high-fat control diet was lentil
(5-fold) > common bean (3.9-fold) > chickpea (2.9-fold) > dry pea (2.3-fold). For dry pea and chickpea,
there are over 10,000 genetic variants of these pulses in various breeding programs and germplasm
collections around the world [85]. Knowledge of the prebiotics that account for health-beneficial
effects would provide an opportunity to guide development of cultivars of these crops that have more
favorable gut health properties than those studied in this experiment.
Since differences in microbial populations were observed when total dietary fiber levels were
similar, it suggests an effect due to some aspect of dietary fiber quality. Because small molecules are
known to be associated with dietary fiber, the small molecule profile of the pulses that were fed was
evaluated and differences in composition were observed in classes of chemical compounds that have
limited bioavailability in mammals and that microbes are known to metabolize [86,87]. For example,
lentil was a distinct source of 3-fucosyllactose, which is an oligosaccharide identified in human milk
and that has distinct beneficial effects on the human intestine. Of equal interest was the dominant
presence of gulonic acid, a known substrate for bacteria in the Bifidobacterium genus. It is also likely
that different carbohydrate moieties included within the framework of total dietary fiber vary among
pulses in a manner that supports the propagation of different microbial colonies within the gut.
There is a growing awareness of the value of increasing pulse consumption [1,64], in part
because this will close the dietary fiber gap and decrease the risk for chronic diseases. However, as
we recently reviewed [88], consumers have concerns about food tolerance that need to be addressed.
The level of pulse consumption studied was intentionally high, i.e., approximately twice as high as
median intakes of pulse consuming subgroups in the US and Canada. However, the dietary level was
actually similar to dietary amounts consumed in developing countries where pulses are dietary
staples. Moreover, the level incorporated into the diet (approximately 18g total dietary fiber/1000
kcal) would achieve levels of dietary intake in humans shown to reduce cardiometabolic disease
related mortality by over 50% in the AARP cohort [14]. We examined several markers of intestinal
function in either the ileum and/or the ascending, transverse, and descending segments of the colon.
Crypt height, the rate of cell proliferation and its location within the crypt, and the amount of alcian
blue positive mucin within goblet cells lining the intestinal lumen were assessed. The limited changes
observed that were not statistically significant after adjustment for false discovery. This finding is
consistent with our previous report that cooked pulses are well tolerated because anti-nutrients are
completely inactivated by heat [89]. Our findings of normal gut physiology based on crypt size,
mucus synthesis, and cell proliferation are consistent with the fact that pulse consumption at 2–3
times the intake observed in countries such as the United States and Canada is well tolerated. It is
noteworthy that some laboratories have reported effects on the same intestinal morphometric
parameters and have interpreted those effects as consistent with improved gut integrity. Our findings
do not rebut those findings. Rather, our extensive experience in morphometric analyses recognized
that differences in technical approaches can render different outcomes. In our judgement, the
combined data of a number of publications [19,21–24,72] confirm that pulses are well tolerated and
underscore the value of functional rather than morphometric assessment of gut integrity as an
objective approach to moving the field forward.
Pulse consumption has been reported to improve blood lipid profiles associated with
cardiovascular disease in clinical studies and in preclinical experiments in the model of obesity used
in the work reported herein [42]. One possible contributor to pulse-mediated beneficial effects on
circulating lipids involves changes in bile acid metabolism [89]. Since it has been shown that a shift
in the ratio of Bacterioidetes to Firmicutes alters intestinal bile salt hydrolase activity in the ileum,
and that such changes alter the expression of the bile acid receptor FXR [48,90–92], ileal transcript
levels were assessed. Interestingly, all pulses induced expression of 1.5- to 1.8-fold relative to the
obesogenic control diet. Whether this is enough to induce changes in bile acid metabolism in the liver
Nutrients 2020, 12, 593 12 of 17

associated with health benefits and/or results in changes in lipid metabolism in adipose tissue will
require further detailed investigation. However, the fact that pulse consumption reduced lipid
accumulation in subcutaneous and visceral adipose depots (Table 3) is consistent with mediation by
alterations in bile acid metabolism and receptor driven signaling with FXR being a candidate
transcription factor.
This study has several limitations. They include (1) the inability to fully match chemical
composition across dietary formulations when a whole food (pulse) approach is used; we argue that
this approach is essential for understanding how the consumer may benefit from pulse crop
consumption; (2) pulses were cooked and then immediately freeze dried; the potential impact of this
approach on qualitative changes in carbohydrate constituents is unknown, but this approach is being
used to make commercially available pulse powders that are being used as ingredients in the design
of new food products; and (3) content of specific bacterial populations was determined by qPCR;
while not as much information is obtained about overall microbial ecology as attained via high-
throughput techniques, qPCR is a rapid, specific, and cost effect method [50].

5. Conclusions
Pulses are an affordable and accessible source of dietary protein and fiber. Encouraging the
consumption of pulses as a dietary staple rather than as a vegetable side dish could eliminate the
long-standing dietary fiber gap and result in levels of fiber intake that promote gut health and the
prevention of four interrelated chronic diseases: obesity, type 2 diabetes, cardiovascular disease, and
certain types of cancer in the absence of adverse outcomes. Our finding of differential effects of the
four most commonly consumed, pulses, chickpea, common bean, dry pea and lentil, on specific
health-beneficial commensal microbes residing in the cecum indicates the importance of defining the
key prebiotic components of pulses so that producers can market value added cultivars of each pulse
and consumers can be guided to the preparation and/or purchase of pulse-based foods that maintain
health-beneficial effects. Studies of the impact of these pulses on the microbial ecology of each
segment of the intestinal tract are also needed to fully understand their impact on intestinal function
and to permit the assessment of whether fecal 16s rRNA profiling has merit as preclinical experiments
are extended to clinical investigations.
Supplementary Materials: The following are available online at www.mdpi.com/xxx/s1. Figure S1: Intestinal
tissue in high-fat control, Table S1: Composition of experimental diets, Table S2: Pulse food composition data,
Table S3: qPCR Gene expression primers and bacterial primers, Table S4: Effect of feeding of pulses on intestinal
alcian blue stained mucin, Table S5 Effect of feeding of pulses on intestinal cell proliferation, Table S6:
Metabolites that discriminate pulse types, and Table S7: Metabolite data.

Author Contributions: Conceptualization, J.N.M., E.S.N., V.K.F., T.L.W., R.M., G.V., and H.J.T.; methodology,
V.K.F., T.L.W., A.L.H., and H.M.O.; validation, T.L.W. and A.L.H.; formal analysis, J.N.M., E.S.N., V.K.F., A.L.H.,
and H.J.T.; investigation, H.J.T.; data curation, J.N.M., E.S.N., H.M.O., and H.J.T.; writing—original draft
preparation, J.N.M., E.S.N., and H.J.T.; writing—review and editing, J.N.M., E.S.N., H.J.T., T.L.W., R.M., and
G.V.; supervision, J.N.M., E.S.N., V.K.F., and H.J.T.; project administration, H.J.T.; funding acquisition, H.J.T. All
authors have read and agreed to the published version of the manuscript.

Funding: This research was funded by the Agricultural Research Service, U.S. Department of Agriculture, grant
number 3060-21650-001-08S

Acknowledgments: We thank Barry Ogg, Colorado State University Agricultural Experiment Station for
production of the white kidney (cannellini) beans used in this study. We also thank Angel McKay Whiteman,
Christian Anthony Isham and Madyson Streeter-McDonald, Colorado State University Cancer Prevention
Laboratory for their excellent technical assistance.

Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design of the
study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to
publish the results.
Nutrients 2020, 12, 593 13 of 17

References
1. Curran, J.; McLachlan, M.; Black, R.; Widders, I.; Manary, M. Collaboration among sectors to increase pulse
consumption. Ann. N. Y. Acad. Sci. 2017, 1392, 3–5, doi:10.1111/nyas.13321.
2. Mitchell, D.C.; Lawrence, F.R.; Hartman, T.J.; Curran, J.M. Consumption of dry beans, peas, and lentils
could improve diet quality in the US population. J. Am. Diet. Assoc 2009, 109, 909–913, doi:S0002-
8223(09)00258-2 pii ;10.1016/j.jada.2009.02.029 doi.
3. Fischer, C.G.; Garnett, T. Plates, pyramids and planet. Available online: http://www.fao.org/3/a-i5640e.pdf
(accessed on 4 August 2017).
4. Garnett, T. Changing what we eat. Available online:
http://www.fcrn.org.uk/sites/default/files/fcrn_wellcome_gfs_changing_consumption_report_final.pdf
(accessed on 3 August 2017).
5. FAO. FAO highlights role of pulses in fighting hunger and achieving healthy diets for all. Available online:
http://www.fao.org/news/story/en/item/1260464/icode (accessed on 9 February 2020).
6. Thompson, H.J.; Brick, M.A. Perspective: Closing the Dietary Fiber Gap: An Ancient Solution for a 21st
Century Problem. Adv. Nutr. 2016, 7, 623–626, doi:10.3945/an.115.009696.
7. Burkitt, D.P.; Walker, A.R.P.; Painter, N.S. Effect of Dietary Fiber on Stools and Transit-Times, and Its Role
in Causation of Disease. Lancet 1972, 1, 1408–1411.
8. Cho, S.S.; Qi, L.; Fahey, G.C.; Klurfeld, D.M. Consumption of cereal fiber, mixtures of whole grains and
bran, and whole grains and risk reduction in type 2 diabetes, obesity, and cardiovascular disease. Am. J.
Clin. Nutr. 2013, 98, 594–619, doi:10.3945/ajcn.113.067629.
9. Viguiliouk, E.; Blanco, M.S.; Kendall, C.W.; Sievenpiper, J.L. Can pulses play a role in improving
cardiometabolic health? Evidence from systematic reviews and meta-analyses. Ann. N. Y. Acad. Sci. 2017,
1392, 43–57, doi:10.1111/nyas.13312.
10. Kim, S.J.; de Souza, R.J.; Choo, V.L.; Ha, V.; Cozma, A.I.; Chiavaroli, L.; Mirrahimi, A.; Mejia, S.B.; Di Buono,
M.; Bernstein, A.M., et al. Effects of dietary pulse consumption on body weight: A systematic review and
meta-analysis of randomized controlled trials. Am. J. Clin. Nutr. 2016, 103, 1213–1223,
doi:10.3945/ajcn.115.124677.
11. Broeckling, C.D.; Afsar, F.A.; Neumann, S.; Ben-Hur, A.; Prenni, J.E. RAMClust: A novel feature clustering
method enables spectral-matching-based annotation for metabolomics data. Anal. Chem. 2014, 86, 6812–
6817, doi:10.1021/ac501530d [doi].
12. Ha, V.; Sievenpiper, J.L.; de Souza, R.J.; Jayalath, V.H.; Mirrahimi, A.; Agarwal, A.; Chiavaroli, L.; Mejia,
S.B.; Sacks, F.M.; Di Buono, M., et al. Effect of dietary pulse intake on established therapeutic lipid targets
for cardiovascular risk reduction: A systematic review and meta-analysis of randomized controlled trials.
Can. Med. Assoc. J. 2014, 186, E252–E262, doi:10.1503/cmaj.131727.
13. Sievenpiper, J.L.; Kendall, C.W.C.; Esfahani, A.; Wong, J.M.W.; Carleton, A.J.; Jiang, H.Y.; Bazinet, R.P.;
Vidgen, E.; Jenkins, D.J.A. Effect of non-oil-seed pulses on glycaemic control: A systematic review and
meta-analysis of randomised controlled experimental trials in people with and without diabetes.
Diabetologia 2009, 52, 1479–1495, doi:10.1007/s00125-009-1395-7.
14. Park, Y.; Subar, A.F.; Hollenbeck, A.; Schatzkin, A. Dietary fiber intake and mortality in the NIH-AARP
diet and health study. Arch. Intern. Med. 2011, 171, 1061–1068, doi:10.1001/archinternmed.2011.18.
15. FAO. Definition and classification of commodities: Pulses and derived products. Available online:
http://www.fao.org/es/faodef/fdef04e.htm (accessed on 20 January 2017).
16. Ciudad-Mulero, M.; Fernandez-Ruiz, V.; Matallana-Gonzalez, M.C.; Morales, P. Dietary fiber sources and
human benefits: The case study of cereal and pseudocereals. Adv. Food Nutr. Res. 2019, 90, 83–134,
doi:10.1016/bs.afnr.2019.02.002.
17. Gowd, V.; Xie, L.H.; Zheng, X.D.; Chen, W. Dietary fibers as emerging nutritional factors against diabetes:
Focus on the involvement of gut microbiota. Crit. Rev. Biotechnol. 2019, 39, 524–540,
doi:10.1080/07388551.2019.1576025.
18. Lin, D.; Peters, B.A.; Friedlander, C.; Freiman, H.J.; Goedert, J.J.; Sinha, R.; Miller, G.; Bernstein, M.A.;
Hayes, R.B.; Ahn, J. Association of dietary fibre intake and gut microbiota in adults. Br. J. Nutr. 2018, 120,
1014–1022, doi:10.1017/S0007114518002465.
19. Monk, J.M.; Wu, W.; Lepp, D.; Wellings, H.R.; Hutchinson, A.L.; Liddle, D.M.; Graf, D.; Pauls, K.P.;
Robinson, L.E.; Power, K.A. Navy bean supplemented high-fat diet improves intestinal health, epithelial
Nutrients 2020, 12, 593 14 of 17

barrier integrity and critical aspects of the obese inflammatory phenotype. J. Nutr. Biochem. 2019, 70, 91–
104, doi:10.1016/j.jnutbio.2019.04.009.
20. Tomova, A.; Bukovsky, I.; Rembert, E.; Yonas, W.; Alwarith, J.; Barnard, N.D.; Kahleova, H. The Effects of
Vegetarian and Vegan Diets on Gut Microbiota. Front. Nutr. 2019, 6, doi:ARTN 47 10.3389/fnut.2019.00047.
21. Monk, J.M.; Lepp, D.; Wu, W.Q.; Graf, D.; McGillis, L.H.; Hussain, A.; Carey, C.; Robinson, L.E.; Liu, R.H.;
Asao, R., et al. Chickpea-supplemented diet alters the gut microbiome and enhances gut barrier integrity
in C57Bl/6 male mice. J. Funct. Foods 2017, 38, 663–674, doi:10.1016/j.jff.2017.02.002.
22. Monk, J.M.; Lepp, D.; Wu, W.Q.; Pauls, K.P.; Robinson, L.E.; Power, K.A. Navy and black bean
supplementation primes the colonic mucosal microenvironment to improve gut health. J. Nutr. Biochem.
2017, 49, 89–100, doi:10.1016/j.jnutbio.2017.08.002.
23. Monk, J.M.; Wu, W.Q.; Hutchinson, A.L.; Pauls, P.; Robinson, L.E.; Power, K.A. Navy and black bean
supplementation attenuates colitis-associated inflammation and colonic epithelial damage. J. Nutr. Biochem.
2018, 56, 215–223, doi:10.1016/j.jnutbio.2018.02.013.
24. Monk, J.M.; Zhang, C.P.; Wu, W.Q.; Zarepoor, L.; Lu, J.T.; Liu, R.H.; Pauls, K.P.; Wood, G.A.; Tsao, R.;
Robinson, L.E., et al. White and dark kidney beans reduce colonic mucosal damage and inflammation in
response to dextran sodium sulfate. J. Nutr. Biochem. 2015, 26, 752–760, doi:10.1016/j.jnutbio.2015.02.003.
25. Chen, Y.R.; McGee, R.; Vandemark, G.; Brick, M.; Thompson, H.J. Dietary Fiber Analysis of Four Pulses
Using AOAC 2011.25: Implications for Human Health. Nutrients 2016, 8, doi:ARTN 829 10.3390/nu8120829.
26. Brummer, Y.; Kaviani, M.; Tosh, S.M. Structural and functional characteristics of dietary fibre in beans,
lentils, peas and chickpeas. Food Res. Intern. 2015, 67, 117–125.
27. Tosh, S.M. Review of human studies investigating the post-prandial blood-glucose lowering ability of oat
and barley food products. Eur. J. Clin. Nutr. 2013, 67, 310–317, doi:ejcn201325 [pii];10.1038/ejcn.2013.25
[doi].
28. Tosh, S.M.; Yada, S. Dietary fibres in pulse seeds and fractions: Characterization, functional attributes, and
applications. Food Res. Intern. 2010, 43, 450–460.
29. Proctor, L.; LoTempio, J.; Marquitz, A.; Daschner, P.; Xi, D.; Flores, R.; Brown, L.; Ranallo, R.; Maruvada,
P.; Regan, K., et al. A review of 10 years of human microbiome research activities at the US National
Institutes of Health, Fiscal Years 2007-2016. Microbiome 2019, 7, 31, doi:10.1186/s40168-019-0620-y.
30. Goodrich, J.K.; Di Rienzi, S.C.; Poole, A.C.; Koren, O.; Walters, W.A.; Caporaso, J.G.; Knight, R.; Ley, R.E.
Conducting a microbiome study. Cell 2014, 158, 250–262, doi:10.1016/j.cell.2014.06.037.
31. Kim, D.; Hofstaedter, C.E.; Zhao, C.; Mattei, L.; Tanes, C.; Clarke, E.; Lauder, A.; Sherrill-Mix, S.; Chehoud,
C.; Kelsen, J., et al. Optimizing methods and dodging pitfalls in microbiome research. Microbiome 2017, 5,
52, doi:10.1186/s40168-017-0267-5.
32. Hildebrandt, M.A.; Hoffmann, C.; Sherrill-Mix, S.A.; Keilbaugh, S.A.; Hamady, M.; Chen, Y.Y.; Knight, R.;
Ahima, R.S.; Bushman, F.; Wu, G.D. High-fat diet determines the composition of the murine gut
microbiome independently of obesity. Gastroenterology 2009, 137, 1716–1724 e1711-1712,
doi:10.1053/j.gastro.2009.08.042.
33. Ravussin, Y.; Koren, O.; Spor, A.; LeDuc, C.; Gutman, R.; Stombaugh, J.; Knight, R.; Ley, R.E.; Leibel, R.L.
Responses of gut microbiota to diet composition and weight loss in lean and obese mice. Obesity (Silver
Spring) 2012, 20, 738–747, doi:10.1038/oby.2011.111.
34. Murphy, E.F.; Cotter, P.D.; Healy, S.; Marques, T.M.; O'Sullivan, O.; Fouhy, F.; Clarke, S.F.; O'Toole, P.W.;
Quigley, E.M.; Stanton, C., et al. Composition and energy harvesting capacity of the gut microbiota:
Relationship to diet, obesity and time in mouse models. Gut 2010, 59, 1635–1642,
doi:10.1136/gut.2010.215665.
35. Gu, Y.; Liu, C.; Zheng, N.; Jia, W.; Zhang, W.; Li, H. Metabolic and Gut Microbial Characterization of
Obesity-Prone Mice under a High-Fat Diet. J. Proteome. Res. 2019, 18, 1703–1714,
doi:10.1021/acs.jproteome.8b00945.
36. Serino, M.; Luche, E.; Gres, S.; Baylac, A.; Berge, M.; Cenac, C.; Waget, A.; Klopp, P.; Iacovoni, J.; Klopp, C.,
et al. Metabolic adaptation to a high-fat diet is associated with a change in the gut microbiota. Gut 2012, 61,
543–553, doi:10.1136/gutjnl-2011-301012.
37. Gajda, A.M. High Fat Diets for Diet-Induced Obesity Models. Available online:
http://www.labanimal.co.kr/product/pdf/Obesity.pdf (accessed on 17 February 2020).
38. Slavin, J.L. Dietary fiber and body weight. Nutrition 2005, 21, 411–418, doi:S0899-9007(04)00304-1
[pii];10.1016/j.nut.2004.08.018 [doi].
Nutrients 2020, 12, 593 15 of 17

39. National Research, C. Guide for the Care and Use of Laboratory Animals: Eighth Edition; The National
Academies Press: Washington, DC, USA, 2011.
40. Chu, D.T.; Malinowska, E.; Jura, M.; Kozak, L.P. C57BL/6J mice as a polygenic developmental model of
diet-induced obesity. Physiol. Rep. 2017, 5, doi:10.14814/phy2.13093.
41. Neil, E.S.; McGinley, J.N.; Fitzgerald, V.K.; Lauck, C.A.; Tabke, J.A.; Streeter-McDonald, M.R.; Yao, L.;
Broeckling, C.D.; Weir, T.L.; Foster, M.T., et al. White Kidney Bean (Phaseolus Vulgaris L.) Consumption
Reduces Fat Accumulation in a Polygenic Mouse Model of Obesity. Nutrients 2019, 11, E2780,
doi:10.3390/nu11112780.
42. Zhu, Z.; Jiang, W.; Thompson, H.J. Edible dry bean consumption (Phaseolus vulgaris L.) modulates
cardiovascular risk factors and diet-induced obesity in rats and mice. Br. J. Nutr. 2012, 108 Suppl 1, S66-73,
doi:10.1017/S0007114512000839.
43. Thompson, M.D.; Thompson, H.J.; Brick, M.A.; McGinley, J.N.; Jiang, W.; Zhu, Z.; Wolfe, P. Mechanisms
associated with dose-dependent inhibition of rat mammary carcinogenesis by dry bean (Phaseolus
vulgaris, L.). J. Nutr. 2008, 138, 2091–2097, doi:10.3945/jn.108.094557.
44. Eade, M.N.; Cooke, W.T.; Brooke, B.N.; Thompson, H. LIVER DISEASE IN CROHNS COLITIS - STUDY
OF 21 CONSECUTIVE PATIENTS HAVING COLECTOMY. Annals of Internal Medicine 1971, 74, 518–&.
45. Thompson, H.J.; McGinley, J.N.; Neil, E.S.; Brick, M.A. Beneficial Effects of Common Bean on Adiposity
and Lipid Metabolism. Nutrients 2017, 9, doi:10.3390/nu9090998.
46. Thompson, M.D.; Mensack, M.M.; Jiang, W.; Zhu, Z.; Lewis, M.R.; McGinley, J.N.; Brick, M.A.; Thompson,
H.J. Cell signaling pathways associated with a reduction in mammary cancer burden by dietary common
bean (Phaseolus vulgaris L.). Carcinogenesis 2012, 33, 226–232, doi:10.1093/carcin/bgr247.
47. Bobe, G.; Barrett, K.G.; Mentor-Marcel, R.A.; Saffiotti, U.; Young, M.R.; Colburn, N.H.; Albert, P.S.; Bennink,
M.R.; Lanza, E. Dietary cooked navy beans and their fractions attenuate colon carcinogenesis in
azoxymethane-induced ob/ob mice. Nutr. Cancer. 2008, 60, 373–381, doi:10.1080/01635580701775142.
48. Jiang, C.; Xie, C.; Lv, Y.; Li, J.; Krausz, K.W.; Shi, J.; Brocker, C.N.; Desai, D.; Amin, S.G.; Bisson, W.H., et al.
Intestine-selective farnesoid X receptor inhibition improves obesity-related metabolic dysfunction. Nat.
Commun. 2015, 6, 10166, doi:10.1038/ncomms10166.
49. Livak, K.J.; Schmittgen, T.D. Analysis of relative gene expression data using real-time quantitative PCR
and the 2(-Delta Delta C(T)) Method. Methods 2001, 25, 402–408, doi:10.1006/meth.2001.1262.
50. De Gregoris, T.B.; Aldred, N.; Clare, A.S.; Burgess, J.G. Improvement of phylum- and class-specific primers
for real-time PCR quantification of bacterial taxa. J. Microbiol. Meth. 2011, 86, 351–356,
doi:10.1016/j.mimet.2011.06.010.
51. Collado, M.C.; Derrien, M.; Isolauri, E. Intestinal integrity and Akkermansia muciniphila, a mucin-
degrading member of the intestinal microbiota present in infants, adults, and the elderly. Appl. Environ.
Microb. 2007, 73, 7767–7770, doi:10.1128/Aem.01477-07.
52. Koliada, A.; Syzenko, G.; Moseiko, V.; Budovska, L.; Puchkov, K.; Perederiy, V.; Gavalko, Y.; Dorofeyev,
A.; Romanenko, M.; Tkach, S., et al. Association between body mass index and Firmicutes/Bacteroidetes
ratio in an adult Ukrainian population. BMC Microbiol. 2017, 17, 120, doi:10.1186/s12866-017-1027-1.
53. Luna, L.G. Manual of histologic staining methods of the Armed Forces Institute of Pathology, Third ed.;
McGraw-Hill: New York, NY, USA; 1968; pp. 258.
54. Matthews, S.B.; McGinley, J.N.; Neil, E.S.; Thompson, H.J. Premenopausal Obesity and Breast Cancer
Growth Rates in a Rodent Model. Nutrients 2016, 8, 214, doi:10.3390/nu8040214.
55. Tuominen, V.J.; Ruotoistenmaki, S.; Viitanen, A.; Jumppanen, M.; Isola, J. ImmunoRatio: A publicly
available web application for quantitative image analysis of estrogen receptor (ER), progesterone receptor
(PR), and Ki-67. Breast Cancer Res. 2010, 12, R56, doi:10.1186/bcr2615.
56. Bettenhausen, H.M.; Barr, L.; Broeckling, C.D.; Chaparro, J.M.; Holbrook, C.; Sedin, D.; Heuberger, A.L.
Influence of malt source on beer chemistry, flavor, and flavor stability. Food Res. Int. 2018, 113, 487–504,
doi:10.1016/j.foodres.2018.07.024.
57. Lavergne, F.D.; Broeckling, C.D.; Cockrell, D.M.; Haley, S.D.; Peairs, F.B.; Jahn, C.E.; Heuberger, A.L. GC-
MS Metabolomics to Evaluate the Composition of Plant Cuticular Waxes for Four Triticum aestivum
Cultivars. Int. J. Mol. Sci. 2018, 19, doi:UNSP 249 10.3390/ijms19020249.
58. Broeckling, C.D.; Heuberger, A.L.; Prenni, J.E. Large scale non-targeted metabolomic profiling of serum by
ultra performance liquid chromatography-mass spectrometry (UPLC-MS). J. Vis. Exp. 2013, e50242,
doi:10.3791/50242.
Nutrients 2020, 12, 593 16 of 17

59. Yao, L.X.; Sheflin, A.M.; Broeckling, C.D.; Prenni, J.E. Data Processing for GC-MS- and LC-MS-Based
Untargeted Metabolomics. Methods Mol. Biol. 2019, 1978, 287–299, doi:10.1007/978-1-4939-9236-2_18.
60. Tsugawa, H.; Kind, T.; Nakabayashi, R.; Yukihira, D.; Tanaka, W.; Cajka, T.; Saito, K.; Fiehn, O.; Arita, M.
Hydrogen rearrangement rules: Computational MS/MS fragmentation and structure elucidation using MS-
FINDER software. Anal. Chem. 2016, 88, 7946–7958, doi:10.1021/acs.analchem.6b00770.
61. Lai, Z.; Tsugawa, H.; Wohlgemuth, G.; Mehta, S.; Mueller, M.; Zheng, Y.; Ogiwara, A.; Meissen, J.;
Showalter, M.; Takeuchi, K., et al. Identifying metabolites by integrating metabolome databases with mass
spectrometry cheminformatics. Nat. Methods 2018, 15, 53–56, doi:10.1038/nmeth.4512.
62. Hummel, J.; Selbig, J.; Walther, D.; Kopka, J. The Golm Metabolome Database: A database for GC-MS based
metabolite profiling. In Metabolomics, Nielsen, J., Jewett, M.C., Eds. Springer: Berlin, Heidelberg, Germany
2007; pp. 75–95.
63. Cleries, R.; Galvez, J.; Espino, M.; Ribes, J.; Nunes, V.; de Heredia, M.L. BootstRatio: A web-based statistical
analysis of fold-change in qPCR and RT-qPCR data using resampling methods. Comput. Biol. Med. 2012, 42,
438–445, doi:10.1016/j.compbiomed.2011.12.012.
64. Havemeier, S.; Erickson, J.; Slavin, J. Dietary guidance for pulses: The challenge and opportunity to be part
of both the vegetable and protein food groups. Ann. N. Y. Acad. Sci. 2017, 1392, 58–66,
doi:10.1111/nyas.13308.
65. Mudryj, A.N.; Yu, N.; Hartman, T.J.; Mitchell, D.C.; Lawrence, F.R.; Aukema, H.M. Pulse consumption in
Canadian adults influences nutrient intakes. Brit J Nutr 2012, 108, S27-S36, doi:10.1017/S0007114512000724.
66. FAO. International year of pulses: Nutritious seeds for a sustainable future. Available online:
http://www.fao.org/3/a-mr021e.pdf (accessed on 9 December 2016).
67. Kleintop, A.E.; Echeverria, D.; Brick, L.A.; Thompson, H.J.; Brick, M.A. Adaptation of the AOAC 2011.25
Integrated Total Dietary Fiber Assay To Determine the Dietary Fiber and Oligosaccharide Content of Dry
Edible Beans. J. Agr. Food Chem. 2013, 61, 9719–9726, doi:10.1021/jf403018k.
68. FAO. CODEX Guidelines on nutrition labeling CAC/GL-2-1985 as last ammended 2010. Available online:
http://www.fao.org/ag/humannutrition/33309-01d4d1dd1abc825f0582d9e5a2eda4a74.pdf (accessed on 4
September 2014).
69. McCleary, B.V.; DeVries, J.W.; Rader, J.I.; Cohen, G.; Prosky, L.; Mugford, D.C.; Champ, M.; Okuma, K.
Determination of Insoluble, Soluble, and Total Dietary Fiber (CODEX Definition) by Enzymatic-
Gravimetric Method and Liquid Chromatography: Collaborative Study. J. Aoac. Int. 2012, 95, 824–844,
doi:10.5740/jaoacint.CS2011_25.
70. Jones, J.M. CODEX-aligned dietary fiber definitions help to bridge the 'fiber gap'. Nutr. J. 2014, 13, doi:Artn
34 10.1186/1475-2891-13-34.
71. Jones, J.M. Dietary Fiber Future Directions: Integrating New Definitions and Findings to Inform Nutrition
Research and Communication. Adv. Nutr. 2013, 4, 8–15, doi:10.3945/an.112002907.
72. Monk, J.M.; Lepp, D.; Zhang, C.P.; Wu, W.; Zarepoor, L.; Lu, J.T.; Pauls, K.P.; Tsao, R.; Wood, G.A.;
Robinson, L.E., et al. Diets enriched with cranberry beans alter the microbiota and mitigate colitis severity
and associated inflammation. J. Nutr. Biochem. 2016, 28, 129–139.
73. Maier, E.; Anderson, C.R.; Roy, C.N. Understanding How Commensal Obligate Anaerobic Bacteria
Regulate Immune Functions in the Large Intestine. Nutrients 2015, 7, doi:10.3390/nu7010045.
74. Caesar, R.; Tremaroli, V.; Kovatcheva-Datchary, P.; Cani, P.D.; Backhed, F. Crosstalk between Gut
Microbiota and Dietary Lipids Aggravates WAT Inflammation through TLR Signaling. Cell Metab. 2015, 22,
658–668, doi:10.1016/j.cmet.2015.07.026.
75. Centre, W.U.a.R. Intestinal bacterium Akkermansia curbs obesity. Available online:
https://www.sciencedaily.com/releases/2013/05/130515113744.htm (accessed on 14 September 2019).
76. Dao, M.; Everard, A.; Aron-Wisnewsky, J.; Sokolovska, N.; Verger, E.; Rizkalla, S.; Dore, J.; Cani, P.; Clenent,
K. Akkermansia muciniphila and Gut Microbiota Richness are Associated with Improved Metabolic Status
after Calorie Restriction. Faseb. J. 2015, 29, 601–603.
77. Derrien, M.; Belzer, C.; de Vos, W.M. Akkermansia muciniphila and its role in regulating host functions.
Microb. Pathogenesis 2017, 106, 171–181, doi:10.1016/j.micpath.2016.02.005.
78. Everard, A.; Belzer, C.; Geurts, L.; Ouwerkerk, J.P.; Druart, C.; Bindels, L.B.; Guiot, Y.; Derrien, M.;
Muccioli, G.G.; Delzenne, N.M., et al. Cross-talk between Akkermansia muciniphila and intestinal
epithelium controls diet-induced obesity. P. Natl. Acad. Sci. USA 2013, 110, 9066–9071,
doi:10.1073/pnas.1219451110.
Nutrients 2020, 12, 593 17 of 17

79. van der Lugt, B.; van Beek, A.A.; Aalvink, S.; Meijer, B.; Sovran, B.; Vermeij, W.P.; Brandt, R.M.C.; de Vos,
W.M.; Savelkoul, H.F.J.; Steegenga, W.T., et al. Akkermansia muciniphila ameliorates the age-related
decline in colonic mucus thickness and attenuates immune activation in accelerated aging Ercc1(-/7) mice.
Immun Ageing 2019, 16, doi:10.1186/s12979-019-0145-z.
80. Depommier, C.; Everard, A.; Druart, C.; Plovier, H.; Van Hul, M.; Vieira-Silva, S.; Falony, G.; Raes, J.;
Maiter, D.; Delzenne, N.M., et al. Supplementation with Akkermansia muciniphila in overweight and obese
human volunteers: A proof-of-concept exploratory study. Nat. Med. 2019, 25, 1096–1103,
doi:10.1038/s41591-019-0495-2.
81. Rinninella, E.; Cintoni, M.; Raoul, P.; Lopetuso, L.R.; Scaldaferri, F.; Pulcini, G.; Miggiano, G.A.D.;
Gasbarrini, A.; Mele, M.C. Food Components and Dietary Habits: Keys for a Healthy Gut Microbiota
Composition. Nutrients 2019, 11, doi:10.3390/nu11102393.
82. Rinninella, E.; Mele, M.C.; Merendino, N.; Cintoni, M.; Anselmi, G.; Caporossi, A.; Gasbarrini, A.; Minnella,
A.M. The Role of Diet, Micronutrients and the Gut Microbiota in Age-Related Macular Degeneration: New
Perspectives from the Gut(-)Retina Axis. Nutrients 2018, 10, doi:10.3390/nu10111677.
83. Rinninella, E.; Raoul, P.; Cintoni, M.; Franceschi, F.; Miggiano, G.A.D.; Gasbarrini, A.; Mele, M.C. What is
the Healthy Gut Microbiota Composition? A Changing Ecosystem across Age, Environment, Diet, and
Diseases. Microorganisms 2019, 7, doi:10.3390/microorganisms7010014.
84. Mariat, D.; Firmesse, O.; Levenez, F.; Guimaraes, V.; Sokol, H.; Dore, J.; Corthier, G.; Furet, J.P. The
Firmicutes/Bacteroidetes ratio of the human microbiota changes with age. BMC Microbiol. 2009, 9, 123,
doi:10.1186/1471-2180-9-123.
85. Joshi, P.K.; Rao, P.P. Global pulses scenario: Status and outlook. Ann. N. Y. Acad. Sci. 2017, 1392, 6–17,
doi:10.1111/nyas.13298.
86. Zmora, N.; Suez, J.; Elinav, E. You are what you eat: Diet, health and the gut microbiota. Nat Rev
Gastroenterol. Hepatol. 2019, 16, 35–56, doi:10.1038/s41575-018-0061-2.
87. Sheflin, A.M.; Melby, C.L.; Carbonero, F.; Weir, T.L. Linking dietary patterns with gut microbial
composition and function. Gut Microbes 2017, 8, 113–129, doi:10.1080/19490976.2016.1270809.
88. Thompson, H.J. Improving human dietary choices through understanding of the tolerance and toxicity of
pulse crop constituents. Current Opinion Food Sci. 2019, 10.1016/j.cofs.2019.01.001,
doi:10.1016/j.cofs.2019.01.001.
89. Daniell, E.L.; Ryan, E.P.; Brick, M.A.; Thompson, H.J. Dietary dry bean effects on hepatic expression of
stress and toxicity-related genes in rats. Br J Nutr 2012, 108 Suppl 1, S37-45, doi:10.1017/S0007114512000815.
90. Gonzalez, F.J.; Jiang, C.; Patterson, A.D. An Intestinal Microbiota-Farnesoid X Receptor Axis Modulates
Metabolic Disease. Gastroenterology 2016, 151, 845–859.
91. Gonzalez, F.J.; Jiang, C.; Xie, C.; Patterson, A.D. Intestinal Farnesoid X Receptor Signaling Modulates
Metabolic Disease. Dig. Dis 2017, 35, 178–184.
92. Pathak, P.; Cen, X.; Nichols, R.G.; Ferrell, J.M.; Boehme, S.; Krausz, K.W.; Patterson, A.D.; Gonzalez, F.J.;
Chiang, J.Y.L. Intestine farnesoid X receptor agonist and the gut microbiota activate G-protein bile acid
receptor-1 signaling to improve metabolism. Hepatology 2018, doi:10.1002/hep.29857.

© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).
Another random document with
no related content on Scribd:
 Chapter XI
I left the hospital before Peter. My injuries, indeed, were of so slight a
nature that I was confined only a few days, while his were so serious
that the physicians despaired of his life, and he was forced to keep
to his bed for several months. Following my early discharge, I made
daily visits of inquiry to the hospital but it was not until June, 1914,
that I was assured that he would recover. With this good news, came
a certain sense of relief, and I made plans for another voyage to
Europe. The incidents of that voyage—I was in Paris at the
beginning of the war—are of sufficient interest so that I may recount
them in another place, but they bear no relationship to the present
narrative.
Subsequent to his recovery, I have learned since from the physician
who attended him during his protracted illness, Peter returned to
Toledo with his mother. It is probable that he made further literary
experiments. It has even occurred to me that the pivot of his being,
the explanation for his whole course of action may have escaped
me. Although, from the hour of our first meeting, my interest in and
my affection for Peter were deep, assuredly I never imagined that I
should be writing down the history of his life. For the greater part of
the term of our friendship, indeed, I was a writer only in a very
modest sense. I was not on the lookout for the kind of "copy" his
affairs and ideas offered, for at this period I was a reporter of music
and the drama. Even later, when I began to set down my thoughts in
what is euphemistically called a more permanent form, the notion of
using Peter as a subject never presented itself to me, and if he had
asked me to do so during his lifetime, urging me to put aside a pile of
unfinished work in his behalf, the request would have astounded me.
I made, therefore, no special effort to ferret out his secrets. When it
was convenient for both of us we met and, largely by accident, I was
a silent witness of three of his literary experiments. How many others
he may have made, I do not know. It is possible that at some time or
other he may have been inspired by the religious school, the Tolstoy
theory of art, or he may have followed the sensuous lead of Gozzoli
and Debussy, artists whose work intrigued him enormously, or in
another æsthetic avatar, he may have believed that true art is
degrading or coldly classic. There is even the possibility, by no
means remote, that he may have fallen under the influence of the
small-town and psychoanalytic schools. Except in a general way,
however, in a conversation which I shall record at the end of this
chapter, he never mentioned further experiments. It is possible that
others may have evidence bearing on this point. Martha Baker might
make a good witness, but she died in 1911. Mrs. Whiffle knew
nothing of any importance whatever about her son. Since his death I
have interrogated her in vain. She was, indeed, very much
astonished at the little I told her and she will read this book, I think,
with real amazement. The report of Clara Barnes, too, was
negligible. Edith Dale has supplied me with a few facts which I have
inserted where they chronologically belong. Most of my other friends,
Phillip Moeller, Alfred Knopf, Edna Kenton, Pitts Sanborn, Avery
Hopwood, Freddo Sides, Joseph Hergesheimer, even my wife, Fania
Marinoff, never met Peter. Louis Sherwin walked up Fifth Avenue
with us one day, but Peter was unusually silent and after he had left
us at the corner of Fifty-seventh Street, Louis was not sufficiently
curious to ask any questions concerning him. I doubt if Louis could
even recall the incident today. I have inserted advertisements in the
Paris, New York, and Toledo newspapers, begging any one with
pertinent facts or letters in his possession to communicate with me,
but as yet I have received no replies. I have never seen a
photograph of my friend and his mother informs me that she doubts
if he ever sat for one.
The record, therefore, of Peter's literary life, at the conclusion of this
chapter, will be as complete as I can make it. I have tried to set down
the truth as I saw it, leaving out nothing that I remember, even at the
danger of becoming unnecessarily garrulous and rambling. I have
written down all I know because, after all, I may have misunderstood
or misinterpreted, and some one else, with the facts before him, may
be better able to reconstruct the picture of this strange life.
Our next meeting occurred in January, 1919, and his first remark
was, Thank God, you're not shot up! From that time, until the day of
his death, nearly a year later, Peter never mentioned the war to me
again, although I saw him frequently enough, nor did he speak of his
writing, save once, on an occasion which shall be reported in its
proper place.
When we came together for the first time, after the long interval—he
had just returned to New York from Florida—I was surprised at and
even shocked by the purely physical change, which, to be sure, had
a psychical significance, for his face had grown more spiritual. He
had always been slender, but now he was thin, almost emaciated. To
describe his appearance a little later, I might use the word haggard.
His coat, which once fitted his figure snugly, rather hung from his
shoulders. There were white patches in the blue-black of his hair,
deep circles under his eyes, and hollows in his cheeks. But his eyes,
themselves, seemed to shine with a new light, seemed to see
something which I could not even imagine. He had rid himself of
many excrescences and externalities, the purely adscititious
qualities, charming though they might be, which masked his
personality. He had, indeed, discovered himself, although I never
knew how clearly until our last conversation. Peter, without
appearing to be particularly aware of it, had become a mystic. His
emancipation had come through suffering. He was quieter, less
restless, less excitable, still enthusiastic, but with more balance,
more—I do not wish to be misunderstood—irony. He had found life
very satisfying and very hard, very sweet, with something of a bitter
after-taste. He seemed almost holy to me, reminding me at times of
those ascetic monks who crawl two thousand miles on their bellies to
worship at some shrine, or of those Hindu fakirs who lie in one
tortured position for years, their bodies slowly consuming, while their
souls gain fire. That he was ill, very ill, I surmised at once, although,
like everything else I have noted here, this was an impression. He
made no admissions, never spoke of his malady; indeed, for Peter,
he talked astonishingly little about himself. He was pathetic and at
the same time an object for admiration.
Afterwards, I learned from his mother that he suffered from an
incurable disease, the disease that killed him late in 1919. But he
never spoke of this to me and he never complained, unless his
occasional confession that he was tired might be construed as a
complaint.
We had fine times together, of a new kind. The tables, in a sense,
were turned. I had become the writer, however humble, and his
ambition had not been realized. His sympathy with my work, with
what I was trying to do, which he saw almost immediately, saw,
indeed, in the beginning, more clearly than I saw it myself, was
complete. He was never weary of talking about it, at any rate he
never showed me that he was weary, and naturally this drew us very
closely together, for an author is fondest of those men who talk the
most about his work. But this is not the place to publish his opinions
of me, although some of them were so curious and far-seeing—they
were not all flattering by any means—that I shall undoubtedly recur
to them in my autobiography. Fortunately for me, his sympathy grew
as my work progressed, and it seemed amazing to me later, looking
over the book after a period of years, that he had found anything
pleasant to report of Music After the Great War. He had, indeed,
seen something in it, and when I recalled what he had said it was
impossible to feel that he had overstated the case in the interests of
friendship. He had seen the germ, the root of what was to come; he
had seen a suggestion of a style, undeveloped ideas, which he felt
would later be developed, as indeed, to a limited extent, they were.
His plea, to put it concisely, had been for a more personal
expression. He was always asking me, after this or that remark or
anecdote in conversation, why I did not write it, just as I had said it or
told it, and it was a great pleasure for him to perceive in The Merry-
Go-Round and In the Garret (of which he read the proofs just before
his death) some signs of growth in this direction.
You are becoming freer, he would say. You are loosening your
tongue; your heart is beating faster. In time you may liberate those
subconscious ideas which are entangled in your very being. It is only
your conscious self that prevents you from becoming a really
interesting writer. Let that once be as free as the air and the other
will be free too. You must walk boldly and proudly and without fear.
You must search the heart; the mind is negligible in literature as in all
other forms of art. Try to write just as you feel and you will discover
that your feeling is greater than your knowledge of it. The words that
appear on the paper will at first seem strange to you, almost like
hermetic symbols, and it is possible that in the course of time you will
be able to say so much that you yourself will not understand what
you are writing. Do not be afraid of that. Let the current flow freely
when you feel that it is the true current that is flowing.
That is the lesson, he continued, that the creative or critical artist can
learn from the interpreter, the lesson of the uses of personality. The
great interpreters, Rachel, Ristori, Mrs. Siddons, Duse, Bernhardt,
Réjane, Ysaye, Paderewski, and Mary Garden are all big, vibrant
personalities, that the deeper thing, call it God, call it IT, flows
through and permeates. You may not believe this now, but I know it
is true, and you will know it yourself some day. And if you cannot
release your personality, what you write, though it be engraved in
letters an inch deep on stones weighing many tons, will lie like snow
in the street to be melted away by the first rain.
We talked of other writers. Peter drew my attention, for instance, to
the work of Cunninghame Graham, that strange Scotch mystic who
turned his back on civilization to write of the pampas, the arid plains
of Africa, India, and Spain, only to find irony everywhere in every
work of man. But, observed Peter, he could not hate civilization so
intensely had he not lived in it. It is all very well to kick over the
ladder after you have climbed it and set foot on the balcony. Like all
lovers of the simple life, he is very complex. And we discussed
James Branch Cabell, who, Peter told me, was originally a
"romantic." He wrote of knights and ladyes and palfreys with
sympathetic picturesqueness. Of late, however, continued Peter, he,
too, seems to have turned over in bed. Romanticism still appears in
his work but it is undermined by a biting and disturbing irony. He
asks: Are any of the manifestations of modern civilization worthy of
admiration? and like Graham, he seems to answer, No. It is possible
that the public disregard for his earlier and simpler manner may have
produced this metamorphosis. Many a man has become bitter with
less reason. Then he spoke of the attributed influence of Maurice
Hewlett and Anatole France on the work of Cabell. Bernard Shaw,
said Peter, once lost all patience with those critics who insisted that
he was a son of Ibsen and Nietzsche and asserted that it was their
ignorance that prevented them from realizing the debt he owed to
Samuel Butler. Cabell might, with justice, voice a similar complaint,
for if he ever had a literary father it was Arthur Machen. In that
author's The Chronicle of Clemendy, issued in 1888, may be
discovered the same confusion of irony and romance that is to be
traced in the work of Cabell. Moreover, like The Soul of Melicent, the
book purports to be a translation from an old chronicle. I might
further speak of the relationship between Hieroglyphics and Beyond
Life, The Hill of Dreams and The Cream of the Jest, although in each
case the treatment and the style are entirely dissimilar. Machen even
preceded Cabell in his use of unfavourable reviews (Vide the
advertising pages of Beyond Life) in his preface to the 1916 edition
of The Great God Pan. Perhaps, added Peter, Cabell has also read
Herman Melville's Mardi to some advantage. But he is no plagiarist; I
am speaking from the point of view of literary genealogy. Peter, at
my instigation, read a novel or two of Joseph Hergesheimer's. Linda
Condon, he reported, is as evanescent as the spirit of God. Only
those who have encountered Lady Beauty among the juniper trees in
the early dawn will feel this book, and only those who feel will
understand. For Hergesheimer has worked a miracle; he has
brought marble to life, created a vibrant chastity. He has described
ice in words of flame!
One night, quite accidentally, we saw the name of Clara Barnes on a
poster in front of the Metropolitan Opera House. She was singing the
rôle of the Priestess in Aida. We purchased two general admission
tickets and slipped in to hear her. The Priestess, those who have
heard Aida will remember, officiates in the temple scene of the first
act but, like the impersonator of the Bird in Siegfried, she is invisible.
Clara's voice sounded tired and worn, as indeed, it should sound
after those long years of study.
We must go back to see her, Peter urged.
We found a changed and broken Clara. She was dressing alone, but
on the third floor, and the odour of Cœur de Jeannette persisted.
She burst into tears when she saw us.
I can't do it, she moaned. Why did you ever come? I can't do it. I can
only sing with my music in front of me. I shall never be able to sing a
part which appears and there are so few rôles in opera, which permit
you to sing back of the scenery! I can't remember. Now she was
wailing. As fast as I learn one part I forget another.
As we walked away on Fortieth Street, Peter began to relate an
incident he had once read in Plutarch; There was a certain magpie,
belonging to a barber at Rome, which could imitate any word he
heard. One day, a company of passing soldiers blew their trumpets
before the shop and for the next forty-eight hours the magpie was
not only mute but also pensive and melancholy. It was generally
believed that the sound of the trumpets had stunned the bird and
deprived it of both voice and hearing. It appeared, however, that this
was not the case for, says Plutarch, the bird had all the time been
occupied in profound meditation, studying how to imitate the sound
of the trumpets, and when at last master of the trick, he astonished
his friends by a perfect imitation of the flourish on those instruments
it had heard, observing with the greatest exactness all the
repetitions, stops, and changes. This lesson, however, had
apparently been learned at the cost of the whole of its intelligence,
for it made it forget everything it had learned before.
We visited many out-of-the-way places together, Peter and I, the
Negro dance-halls near 135th Street, and the Italian and the Yiddish
Theatres. Peter once remarked that he enjoyed plays more in a
foreign language with which he was unfamiliar. What he could
imagine of plot and dialogue far transcended the actuality. We often
dined at a comfortable Italian restaurant on Spring Street, on the
walls of which birds fluttered through frescoed arbours, trailing with
fruits and flowers, and where the spaghetti was too good to be eaten
without prayer. In an uptown café, we had a strange adventure with a
Frenchwoman, La Tigresse, which I have related elsewhere.[4] Peter
refused, in these last months, to go to concerts, especially in
Carnegie Hall, the atmosphere of which, he said, made it impossible
to listen to music. The bare walls, the bright lights, the sweating
conductors, and the silly, gaping crowd oppressed his spirit. He
envied Ludwig of Bavaria who could listen to music in a darkened
hall in which he was the only auditor. Conditions were more
favourable in the moving picture theatres. The bands, perhaps, did
not play so well but the auditoriums were more subtly lighted, so that
the figures of the audience did not intrude.
Peter was more of a recluse than ever. It had been impossible to
persuade him to meet anybody since the Edith Dale days (Edith
herself was now living in New Mexico and, owing to a slight
misunderstanding, I had not seen or heard from her in five years).
He was even sensitive and morbid on the subject. He made me
promise, as a matter of fact, after the Louis Sherwin episode, that in
case we encountered any of my friends in a restaurant or at a
theatre, I would not introduce him. There was, I assured myself, a
good reason for this. In these last days, Peter faded out in a crowd.
He lost a good deal of his personality even in the presence of a third
person. I begged him to go with me to Florine Stettheimer's studio to
see her pictures, which I was sure would please him, but he refused.
He liked to stroll around with me in odd places and he read and
played the piano a good deal, but he seemed to have few other
interests. He was absolutely ignorant of such matters as politics and
government. He never voted and I have heard him refer to the
president, and not in jest, as Abraham Wilson. Sports did not amuse
him either, but occasionally we went together to see the wrestlers at
Madison Square Garden, especially when Stanislaus Zbyszko was
announced to appear.
He never went to Europe again although, shortly before he died, he
talked of a voyage to Spain. He visited his mother at Toledo several
times and he had planned a trip to Florida, the climate of which he
found particularly soothing to his malady, in January, 1920.
Occasionally he just disappeared, returning again, somewhat
mysteriously, without any explanation, without, indeed, any
admission that he had been away. I knew him too well to ask
questions and, to say truth, there was something very sweet about
these little mystifications. Privacy was so dear a privilege to him that
even with his nearest friends, of which, assuredly, I was one,
perhaps the nearest in this last year, it was essential to his
happiness that he should maintain a certain restraint, a certain
reserve, I had almost said, a certain mystery, but, curiously, there
was nothing theatrical about Peter, even in his most theatrical
performances. Just as by the fineness of his taste, Rembrandt
softened the hideousness of a lurid subject in his Anatomy Lesson,
so the exquisite charm of Peter's personality overcame any possible
repugnance to anything he might choose to do.
During this last year in New York, he lived in an old house on
Beekman Place, that splendid row, just two blocks long, of mellow
brown-stone dwellings, with flights of steps, which back upon the
East River at Fiftieth Street. We often sat on the balcony, looking
over towards the span of the Queensboro Bridge, Blackwell's Island,
with its turreted and battlemented castles so like the Mysteries of
Udolpho, watching the gulls sweep over the surface of the water, the
smoke wreathe from the factory chimneys, and the craft on the river,
with cargoes "of Tyne coal, road-rails, pig-lead, fire-wood, iron-ware,
and cheap tin trays," of the city, but seemingly away from it, with our
backs to it, literally, indeed, while life ebbed by. And, at my side, too,
I saw it slowly ebbing.
The interior, one of those fine old New York interiors, with high
ceilings, bordered with plaster guilloches, white carved marble fire-
places, sliding doors, and huge crystal chandeliers, whose pendants
jingled when some one walked on the floor above, it had been his
happy fancy to decorate in the early Victorian manner. The furniture,
to be sure, was mostly Chippendale, Sheraton, and Heppelwhite, but
there were also heavy carved walnut chairs, upholstered in lovely
figured glazed chintzes. The mirrors were framed in four inches of
purple and red engraved glass. The highboys were littered with
ornaments, Staffordshire china dogs and shepherdesses, splendid
feather and shell flowers, and ormolu clocks stood under glass bells
on the mantel-shelves. He had found a couple of rather worn, but still
handsome, Aubusson carpets, with garlands of huge roses of a pale
blush colour. One of these was in the drawing-room, the other in the
library. An old sampler screen framed the fire-place in the latter
room. The books were curious. Peter was now interested in byways
of literature. I remember such volumes as Thomas Mann's Der Tod
in Venedig, Paterne Berrichon's Life of Arthur Rimbaud, Alfred
Jarry's Ubu Roi, with music by Claude Terrasse, Jean Lorrain's La
Maison Philibert, Richard Garnett's The Twilight of the Gods, the
Comte de Lautréamont's Les Chants de Maldoror, Leolinus
Siluriensis's The Anatomy of Tobacco, Binet-Valmer's Lucien,
Haldane MacFall's The Wooings of Jezebel Pettyfer, James Morier's
Hajji Baba of Ispahan, Robert Hugh Benson's The Necromancers,
André Gide's L'Immoraliste, and various volumes by Guillaume
Apollinaire. The walls of the drawing-room were hung with a French
eighteenth century, rose cotton print, the design of which showed, on
one side, Cupid rowing lustily, while listless old Time sat in the bow
of the boat, with the motto: l'Amour fait passer le Temps; and, on the
other side, Time propelling the boat, while a saddened Cupid, his
face buried in his hands, was the downcast passenger, with the
motto: Le Temps fait passer l'Amour. In the centre, beside a
charming Greek temple, a nymph toyed with a spaniel, and the motto
read: l'Amitié ne craint pas le Temps! There were, therefore, no
pictures on these walls, but, elsewhere, where the walls were white,
or where they were hung with rich crimson Roman damask, as in the
library, there were a few steel engravings and mezzotints and an
early nineteenth century lithograph or two. Over his night-table, at
the side of his bed, he had pinned a photograph of a detail of
Benozzo Gozzoli's frescoes in the Palazzo Riccardi, the detail of the
three youths, and there was also a large framed photograph of
Cranach's naïve Venus in this room. The piano stood in the drawing-
room, near one of the windows, looking over the river. It was always
open and the rack was littered with modern music: John Ireland's
London Pieces, Béla Bartok's Three Burlesques, Gerald Tyrwhitt's
Three Little Funeral Marches, music by Erik Satie, Darius Milhaud,
Georges Auric, and Zoltan Kodaly. I remember one day he asked me
to look at Theodor Streiche's Sprüche and Gedichte, with words by
Richard Dehmel, the second of which he averred was the shortest
song ever composed, consisting of but four bars.
It was a lovely house to lie about in, to talk in, to dream in. It was
restful and quaint, offering a pleasing contrast to the eccentric
modernity of the other homes I visited at this period. There was no
electricity. The chandeliers burned gas but the favourite illumination
was afforded by lamps with round glass globes of various colours,
through which the soft light filtered.
On an afternoon in December, 1919, we were lounging in the
drawing-room. Peter had curled himself into a sort of knot on a broad
sofa with three carved walnut curves at the back. He had spread a
knitted coverlet over his feet, for it was a little chilly, in spite of the
fact that a wood fire was smouldering in the grate. On the table
before him there was a highball glass, half-full of the proper
ingredients, and sprawling beside him on the sofa, a magnificent
blue Persian cat, which he called Chalcedony. George Moore and
George Sand had long since perished of old age and Lou Matagot
had been a victim of the laboratory explosion. There was a certain
melancholy implicit in their absence. Nothing reminds us so
irresistibly of the passing of time as the short age allotted on this
earth to our dear cats. The pinchbottle and several bottles of soda, a
bowl of cracked ice and a bowl of Fatima cigarettes, which both of us
had grown to prefer, reposed conveniently on the table between us. I
remember the increasing silence as the twilight fell and, how, at last,
Peter began to talk.
I wanted to do so much, he began, and for a long time, during these
past four years, it seemed to me that I had done so little. I
remembered Zola's phrase: Mon œuvre, alors, c'était l'Arche, l'Arche
immense! Hélas! ce que l'on rêve, et puis, après, ce que l'on
exécute! At the beginning of the war, I was so very miserable, so
unhappy, so alone. It seemed to me that I had been a complete
failure, that I had accomplished nothing....
I must have raised a protesting hand, for he interjected, No, don't
interrupt me. I am not complaining or asking for sympathy. I am
explaining how I felt, not how I feel. I never spoke of it, of course,
while I felt that way. I am only talking about it now because I have
gone beyond, because, in a sense, at least, I understand. I am
happier now, happier, perhaps, than I have ever been before, for in
the past four years I have left behind my restlessness and achieved
something like peace. I no longer feel that I have failed. Of course, I
have failed, but that was because I was attempting to do something
that I had no right to attempt. My cats should have taught me that. It
is necessary to do only what one must, what one is forced by nature
to do. Samuel Butler has said, and how truly, Nothing is worth doing
or well done which is not done fairly easily, and some little deficiency
of effort is more pardonable than any perceptible excess, for virtue
has ever erred rather on the side of self-indulgence than of
asceticism.... And so, in the end, and after all I am still young, I have
learned that I cannot write. Is a little experience too much to pay for
learning to know oneself? I think not, and that is why, now, I feel
more like a success than a failure, because, finally, I do know myself,
and because I have left no bad work. I can say with Macaulay: There
are no lees in my wine. It is all the cream of the bottle....
I have tried to do too much and that is why, perhaps, I have done
nothing. I wanted to write a new Comédie Humaine. Instead, I have
lived it. And now, I have come to the conclusion that that was all
there was for me to do, just to live, as fully as possible. Sympathy
and enthusiasm are something, after all. I must have communicated
at least a shadow of these to the ideas and objects and people on
whom I have bestowed them. Benozzo Gozzoli's frescoes—now,
don't laugh at what I am going to say, because it is true when you
understand it—are just so much more precious because I have loved
them. They will give more people pleasure because I have given
them my affection. This is something; indeed, next to the creation of
the frescoes, perhaps it is everything.
There are two ways of becoming a writer: one, the cheaper, is to
discover a formula: that is black magic; the other is to have the urge:
that is white magic. I have never been able to discover a new
formula; I have worked with the formulæ of other artists, only to see
the cryptogram blot and blur under my hands. My manipulation of the
mystic figures and the cabalistic secrets has never raised the right
demons....
What is there anyway? All expression lifts us further away from
simplicity and causes unhappiness.... Material, scientific expression:
flying-machines, moving pictures, and telegraphy are simply
disturbing. They add nothing valuable to human life. Any novelist
who invokes the aid of science dies a swift death. Zola's novels are
stuffed with theories of heredity but ideas about heredity change
every day. The current craze is for psychoanalytic novels, which are
not half so psychoanalytic as the books of Jane Austen, as posterity
will find out for itself.... Art in this epoch is too self-conscious.
Everybody is striving to do something new, instead of writing or
painting or composing what is natural.... Even the disturbing irony
and pessimism of Anatole France and Thomas Hardy add nothing to
life. We shall be happier if we go back to the beginning....
The great secret is the cat's secret, to do what one has to do. Let IT
do it, let IT, whatever IT is, flow through you. The writer should say,
with Sancho Panza, De mis viñas vengo, no sé nada. Labanne, in Le
Chat Maigre, cries: Art declines in the degree that thought develops.
In Greece, in the time of Aristotle, there were only sculptors. Artists
are inferior beings. They resemble pregnant women; they give birth
without knowing why. And again, to quote my beloved Samuel Butler,
No one understands how anything is done unless he can do it
himself; and even then he probably does not know how he has done
it. I might add that very often he does not know what he has done.
Sterne wrote Tristram Shandy to ridicule his personal enemies.
Dickens began Pickwick to give the artist, Seymour, an opportunity
to draw Cockney sportsmen and he concluded it in high moral
fervour, with the ambition to wipe out bribery and corruption at
elections, unscrupulous attorneys, and Fleet Prison. To Cervantes,
Don Quixote was a burlesque of the high-flown romantic literature of
his period. To the world, it is one of the great romances of all time....
You see, I am beginning to understand why I haven't written, why I
cannot write.... That is why I am unhappy no longer, why I am more
peaceful, why I do not suffer. But, and now a strange, quavering note
sounded in his voice, if I had found a new formula, who knows what I
might have done?
He turned his face away from me towards the back of the sofa. The
cat was purring heavily, almost like the croupy breathing of a child. It
was quite dark outside, and there was no light in the room save for
the flicker that came from the dying embers. There was a long
silence. In trying afterwards to reckon its length, I judged it must
have been fully half an hour before I spoke. It was a noise that broke
the charm of the stillness. The dead end of the log split over the
andirons and fell into the grate.
Peter, I began.
He did not move.
Peter.... I rose and bent over him. The clock struck six. The cat
stirred uneasily, rose, stretched his enormous length; then gave a
faint but alarmingly portentous mew and leaped from the couch.
Peter!
He did not answer me.
April 29, 1921
New York.

FOOTNOTES:
[4] In the Garret.

THE END
 *** END OF THE PROJECT GUTENBERG EBOOK PETER
WHIFFLE ***

Updated editions will replace the previous one—the old editions will
be renamed.

Creating the works from print editions not protected by U.S.

copyright law means that no one owns a United States copyright in
these works, so the Foundation (and you!) can copy and distribute it
in the United States without permission and without paying copyright
royalties. Special rules, set forth in the General Terms of Use part of
this license, apply to copying and distributing Project Gutenberg™
electronic works to protect the PROJECT GUTENBERG™ concept
and trademark. Project Gutenberg is a registered trademark, and
may not be used if you charge for an eBook, except by following the
terms of the trademark license, including paying royalties for use of
the Project Gutenberg trademark. If you do not charge anything for
copies of this eBook, complying with the trademark license is very
easy. You may use this eBook for nearly any purpose such as
creation of derivative works, reports, performances and research.
Project Gutenberg eBooks may be modified and printed and given
away—you may do practically ANYTHING in the United States with
eBooks not protected by U.S. copyright law. Redistribution is subject
to the trademark license, especially commercial redistribution.

START: FULL LICENSE

THE FULL PROJECT GUTENBERG LICENSE
 PLEASE READ THIS BEFORE YOU DISTRIBUTE OR USE THIS WORK

To protect the Project Gutenberg™ mission of promoting the free

distribution of electronic works, by using or distributing this work (or
any other work associated in any way with the phrase “Project
Gutenberg”), you agree to comply with all the terms of the Full
Project Gutenberg™ License available with this file or online at
www.gutenberg.org/license.

Section 1. General Terms of Use and

Redistributing Project Gutenberg™
electronic works
1.A. By reading or using any part of this Project Gutenberg™
electronic work, you indicate that you have read, understand, agree
to and accept all the terms of this license and intellectual property
(trademark/copyright) agreement. If you do not agree to abide by all
the terms of this agreement, you must cease using and return or
destroy all copies of Project Gutenberg™ electronic works in your
possession. If you paid a fee for obtaining a copy of or access to a
Project Gutenberg™ electronic work and you do not agree to be
bound by the terms of this agreement, you may obtain a refund from
the person or entity to whom you paid the fee as set forth in
paragraph 1.E.8.

1.B. “Project Gutenberg” is a registered trademark. It may only be

used on or associated in any way with an electronic work by people
who agree to be bound by the terms of this agreement. There are a
few things that you can do with most Project Gutenberg™ electronic
works even without complying with the full terms of this agreement.
See paragraph 1.C below. There are a lot of things you can do with
Project Gutenberg™ electronic works if you follow the terms of this
agreement and help preserve free future access to Project
Gutenberg™ electronic works. See paragraph 1.E below.
1.C. The Project Gutenberg Literary Archive Foundation (“the
Foundation” or PGLAF), owns a compilation copyright in the
collection of Project Gutenberg™ electronic works. Nearly all the
individual works in the collection are in the public domain in the
United States. If an individual work is unprotected by copyright law in
the United States and you are located in the United States, we do
not claim a right to prevent you from copying, distributing,
performing, displaying or creating derivative works based on the
work as long as all references to Project Gutenberg are removed. Of
course, we hope that you will support the Project Gutenberg™
mission of promoting free access to electronic works by freely
sharing Project Gutenberg™ works in compliance with the terms of
this agreement for keeping the Project Gutenberg™ name
associated with the work. You can easily comply with the terms of
this agreement by keeping this work in the same format with its
attached full Project Gutenberg™ License when you share it without
charge with others.

1.D. The copyright laws of the place where you are located also
govern what you can do with this work. Copyright laws in most
countries are in a constant state of change. If you are outside the
United States, check the laws of your country in addition to the terms
of this agreement before downloading, copying, displaying,
performing, distributing or creating derivative works based on this
work or any other Project Gutenberg™ work. The Foundation makes
no representations concerning the copyright status of any work in
any country other than the United States.

1.E. Unless you have removed all references to Project Gutenberg:

1.E.1. The following sentence, with active links to, or other

immediate access to, the full Project Gutenberg™ License must
appear prominently whenever any copy of a Project Gutenberg™
work (any work on which the phrase “Project Gutenberg” appears, or
with which the phrase “Project Gutenberg” is associated) is
accessed, displayed, performed, viewed, copied or distributed:
 This eBook is for the use of anyone anywhere in the United
States and most other parts of the world at no cost and with
almost no restrictions whatsoever. You may copy it, give it away
or re-use it under the terms of the Project Gutenberg License
included with this eBook or online at www.gutenberg.org. If you
are not located in the United States, you will have to check the
laws of the country where you are located before using this
eBook.

1.E.2. If an individual Project Gutenberg™ electronic work is derived

from texts not protected by U.S. copyright law (does not contain a
notice indicating that it is posted with permission of the copyright
holder), the work can be copied and distributed to anyone in the
United States without paying any fees or charges. If you are
redistributing or providing access to a work with the phrase “Project
Gutenberg” associated with or appearing on the work, you must
comply either with the requirements of paragraphs 1.E.1 through
1.E.7 or obtain permission for the use of the work and the Project
Gutenberg™ trademark as set forth in paragraphs 1.E.8 or 1.E.9.

1.E.3. If an individual Project Gutenberg™ electronic work is posted

with the permission of the copyright holder, your use and distribution
must comply with both paragraphs 1.E.1 through 1.E.7 and any
additional terms imposed by the copyright holder. Additional terms
will be linked to the Project Gutenberg™ License for all works posted
with the permission of the copyright holder found at the beginning of
this work.

1.E.4. Do not unlink or detach or remove the full Project

Gutenberg™ License terms from this work, or any files containing a
part of this work or any other work associated with Project
Gutenberg™.

1.E.5. Do not copy, display, perform, distribute or redistribute this

electronic work, or any part of this electronic work, without
prominently displaying the sentence set forth in paragraph 1.E.1 with
active links or immediate access to the full terms of the Project
Gutenberg™ License.
1.E.6. You may convert to and distribute this work in any binary,
compressed, marked up, nonproprietary or proprietary form,
including any word processing or hypertext form. However, if you
provide access to or distribute copies of a Project Gutenberg™ work
in a format other than “Plain Vanilla ASCII” or other format used in
the official version posted on the official Project Gutenberg™ website
(www.gutenberg.org), you must, at no additional cost, fee or expense
to the user, provide a copy, a means of exporting a copy, or a means
of obtaining a copy upon request, of the work in its original “Plain
Vanilla ASCII” or other form. Any alternate format must include the
full Project Gutenberg™ License as specified in paragraph 1.E.1.

1.E.7. Do not charge a fee for access to, viewing, displaying,

performing, copying or distributing any Project Gutenberg™ works
unless you comply with paragraph 1.E.8 or 1.E.9.

1.E.8. You may charge a reasonable fee for copies of or providing

access to or distributing Project Gutenberg™ electronic works
provided that:

• You pay a royalty fee of 20% of the gross profits you derive from
the use of Project Gutenberg™ works calculated using the
method you already use to calculate your applicable taxes. The
fee is owed to the owner of the Project Gutenberg™ trademark,
but he has agreed to donate royalties under this paragraph to
the Project Gutenberg Literary Archive Foundation. Royalty
payments must be paid within 60 days following each date on
which you prepare (or are legally required to prepare) your
periodic tax returns. Royalty payments should be clearly marked
as such and sent to the Project Gutenberg Literary Archive
Foundation at the address specified in Section 4, “Information
about donations to the Project Gutenberg Literary Archive
Foundation.”

• You provide a full refund of any money paid by a user who

notifies you in writing (or by e-mail) within 30 days of receipt that
s/he does not agree to the terms of the full Project Gutenberg™
License. You must require such a user to return or destroy all