Carnitine Deficiency in OCTN22/2Newborn Mice Leads
to a Severe Gut and Immune Phenotype with
Widespread Atrophy, Apoptosis and a Pro-Inflammatory
Srinivas Sonne1,2, Prem S. Shekhawat1,2*, Dietrich Matern3, Vadivel Ganapathy2, Leszek Ignatowicz4
1Department of Pediatrics, Medical College of Georgia, Georgia Health Science University, Augusta, Georgia, United States of America, 2Department of Biochemistry and
Molecular Biology, Medical College of Georgia, Georgia Health Sciences University, Augusta, Georgia, United States of America, 3Departments of Laboratory Medicine &
Pathology, Medical Genetics, and Pediatric & Adolescent Medicine, Mayo Clinic College of Medicine, Rochester, Minnesota, United States of America, 4Center for
Biotechnology and Genomic Medicine, Medical College of Georgia, Georgia Health Sciences University, Augusta, Georgia, United States of America
We have investigated the gross, microscopic and molecular effects of carnitine deficiency in the neonatal gut using a mouse
model with a loss-of-function mutation in the OCTN2 (SLC22A5) carnitine transporter. The tissue carnitine content of
neonatal homozygous (OCTN22/2) mouse small intestine was markedly reduced; the intestine displayed signs of stunted
villous growth, early signs of inflammation, lymphocytic and macrophage infiltration and villous structure breakdown.
Mitochondrial b-oxidation was active throughout the GI tract in wild type newborn mice as seen by expression of 6 key
enzymes involved in b-oxidation of fatty acids and genes for these 6 enzymes were up-regulated in OCTN22/2mice. There
was increased apoptosis in gut samples from OCTN22/2mice. OCTN22/2mice developed a severe immune phenotype,
where the thymus, spleen and lymph nodes became atrophied secondary to increased apoptosis. Carnitine deficiency led to
increased expression of CD45-B220+lymphocytes with increased production of basal and anti-CD3-stimulated pro-
inflammatory cytokines in immune cells. Real-time PCR array analysis in OCTN22/2mouse gut epithelium demonstrated
down-regulation of TGF-b/BMP pathway genes. We conclude that carnitine plays a major role in neonatal OCTN22/2mouse
gut development and differentiation, and that severe carnitine deficiency leads to increased apoptosis of enterocytes,
villous atrophy, inflammation and gut injury.
Citation: Sonne S, Shekhawat PS, Matern D, Ganapathy V, Ignatowicz L (2012) Carnitine Deficiency in OCTN22/2Newborn Mice Leads to a Severe Gut and
Immune Phenotype with Widespread Atrophy, Apoptosis and a Pro-Inflammatory Response. PLoS ONE 7(10): e47729. doi:10.1371/journal.pone.0047729
Editor: Markus M. Heimesaat, Charite ´, Campus Benjamin Franklin, Germany
Received June 23, 2012; Accepted September 14, 2012; Published October 24, 2012
Copyright: ? 2012 Sonne et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This study was funded by National Institutes of Health grant HD 048867 to PSS, MD. The funders had no role in study design, data collection and
analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: firstname.lastname@example.org
Neonatal necrotizing enterocolitis (NEC) is a potentially fatal
gastrointestinal emergency of the neonate. It is an acute intestinal
necrosis syndrome of unknown etiology and occurs primarily
among prematurely delivered neonates . Its overall incidence
has been reported to be around 0.3 to 2.4 cases per 1000 live
births and it accounts for 2–5% of all NICU admissions and affects
5–25% of extremely low birth weight infants (,29 weeks gestation)
. Improved standards of care in the NICU have resulted in
more preterm survivors; consequently more cases of this poten-
tially fatal illness are reported . The overall mortality related to
NEC continues to be 20–30% and is inversely proportional to the
gestational age, approaching 40–50% in extremely low birth
weight neonates. NEC, especially in surgical cases leads to
significantly prolonged hospitalization and long-term neurodeve-
lopmental impairment [4,5,6]. Overall up to 45% of NEC
survivors suffer from neurodevelopmental delay and cause
significant increase in health care costs .
Despite several decades of research, the pathogenesis of NEC is
still poorly understood. An interplay of several risk factors, which
include intestinal ischemia-reperfusion injury, enteral feeding, and
poor host defenses leading to sepsis, have been implicated; but
prematurity and feeding are the two accepted, most important risk
factors for this illness [3,8]. Infant formulas have been modified to
reduce osmolality and several additives have been considered to
help reduce NEC occurrence, however, over the years only breast
milk has been proven to be clearly beneficial . Thus nutrition
plays an important role in its causation but the exact role played
by individual macro- and micronutrients is still not delineated.
Carnitine (b-hydroxy c-trimethylaminobutyrate) is a condition-
ally essential nutrient. It is obligatory for transport of long-chain
fatty acids into mitochondria for their subsequent b-oxidation.
Therefore, carnitine plays a critical role in energy metabolism of
tissues that derive a substantial portion of their metabolic energy
from fatty acid oxidation. Such tissues in the past included the
heart, skeletal muscle, liver, and placenta, and recently several
reports have elucidated its role in the GI tract [10,11,12]. The
biological importance of carnitine is underscored by the severe
clinical consequences of carnitine deficiency seen in humans
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Two distinct types of carnitine deficiency have been identified.
Primary carnitine deficiency arises from defects in the plasma
membrane carnitine transporter OCTN2. Patients with this
disorder excrete carnitine in urine due to defective reabsorption,
and plasma and tissue levels of carnitine drop below 10% of
normal [15,16,17,18,19]. These patients have marked defects in
fatty acid oxidation. Secondary carnitine deficiency arises from
defects in any of the enzymes involved in fatty acid oxidation. In
patients with these disorders, organic acids accumulate due to
defective fatty acid oxidation and these organic acids enhance
urinary excretion of carnitine in the form of acylcarnitines [10,20].
Both forms of carnitine deficiency are associated with severe
clinical consequences, notably hypoglycemia, cardiomyopathy,
skeletal myopathy, arrhythmias, neuropathy, fatty liver, and in
some cases sudden unexpected death [21,22].
Most of the studies reported so far on carnitine deficiency in the
neonate focus on the clinical consequences due to dysfunction of
heart and skeletal muscle; little attention has been paid to its effects
on the gastrointestinal tract. Whenever a fatty acid oxidation
disorder (FAOD) is diagnosed, most are promptly treated with
carnitine supplementation, and GI pathology escapes attention of
most clinicians. Carnitine’s role in the GI tract has recently been
highlighted by several publications linking mutations in genes
encoding carnitine transporters OCTN1 (SLC22A4) and OCTN2
(SLC22A5) with Crohn’s disease (CD) [23,24,25,26,27,28,29].
Patients with CD have been shown to have a missense substitution
1762CRT in OCTN1 which causes amino acid substitution
L503F and a GRC transversion in the promoter region of
OCTN2 (2207GRC), which disrupts a heat shock binding
element (HSE) in the promoter region of OCTN2 gene. These
mutations lead to decrease in plasma membrane transport of
carnitine and thus reduce tissue content of carnitine. Carnitine has
also been shown to play a protective role in hypoxia/reoxygena-
tion injury in the neonatal mice [30,31] but there is paucity of
information regarding its effects on developing gut and immune
function in the neonatal GI tract.
Preterm infants are born with 40–50% lower plasma and tissue
levels of carnitine [32,33,34,35]. The normative levels and
postnatal changes in plasma carnitine in the preterm neonate
are now well characterized [36,37,38]. Sick preterm neonates who
do not receive carnitine supplementation via the enteral or
parenteral route continue to have lower tissue carnitine levels
[39,40,41,42] which drop further, and thus may develop a state of
relative tissue carnitine deficiency. We hypothesize that tissue
carnitine deficiency will have an impact at the molecular level in
the developing GI tract. In this report, we have investigated the
role played by carnitine in the neonatal gut using a mouse model
of carnitine deficiency due to defective OCTN2.
Materials and Methods
Animals and Sample Preparation
We obtained a breeding pair of heterozygous OCTN2+/2mice
from Prof. Ikumi Tamai, Kanazawa University, Japan. Heterozy-
gous OCTN2+/2mice are viable and fertile so several pairs of
heterozygous males and females were mated,
pregnant OCTN2+/2mice were allowed to deliver and the pups
were observed closely after birth. Homozygous OCTN22/2and
age-matched wild-type OCTN2+/+mice in the litters were
genotyped as described earlier . Wild type (OCTN2+/+) and
homozygous (OCTN22/2) pups were sacrificed around 7 days of
life to collect tissue samples and to identify the various GI tract and
immune system pathologies evident by this time. The experimen-
tal procedures were approved by the Institutional Animal Care
and Use Committee of the Medical College of Georgia, Georgia
Health Sciences University, GA, USA.
Semi-quantitative RT-PCR for Enzymes Involved in b-
Primers were designed using OligoH primer analysis software
6.0 (National Biosciences Inc. Cascade, CO, USA) for 6 enzymes
involved in b-oxidation of fatty acids. Primer sequences and
amplicon size for each enzyme are as shown in Table-1, all assays
were carried out as described earlier . Densitometry was
performed using a SpectraImager 5000 Imaging system and
AlphaEase 32-bit software (Alpha Innotech, San Leandro, CA,
USA). Each experiment was repeated at least 6 times in each
group from different mouse samples.
Histopathology and Immunohistochemistry of Gut
Mouse gut samples were fixed in 10% formalin and 5–10 mm
thick sections of paraffin-embedded tissue were cut, applied to
glass slides, deparaffinized in xylene, and rehydrated in an ethanol
gradient. A set of sections from 6 animals was stained with
hematoxylin and eosin (H&E) for histological analysis. For
quenched by incubating the specimens in 3% H2O2in methanol
for 30 min and subjected to antigen retrieval.
The slides were then washed and blocked using an Avidin/
Biotin blocking kit (Vector Labs, Burlingame, CA) for 30 min
followed by a blocking buffer (NEN-Life Sciences, Boston, MA) for
30 min. The blocking buffer was removed, and the sections were
exposed to primary rabbit polyclonal antibody specific for each of
the following enzymes at the indicated dilution: MCAD (1:200),
LCAD (1:400), VLCAD (1:200), SCHAD (1:200), LKAT (1:400).
(kind gift of Dr Arnold Strauss, Cincinnati Children’s Hospital,
Table 1. Primers used for RT-PCR studies of FAO enzymes.
Accession NumberEnzyme Amplicon (bp)Sense primer Antisense primer
NM_178878LCHAD520 CAA CGA CCA AAT CAG GAG TGAGA GAC TTT CCG ATC AGC C
NM_007381LCAD576 CCA CTC AGA TAT TGT CAT GCC CACC ATT TCC CCC CCT TTT CC
NM_145558LKAT 564GGA AAG GAC ACA GTT ACC AAA G TGA CAC AGA CAG GAA TAA GGA G
NM_017366VLCAD584 CCA CCA GAG AAA AAC CAG CCAGA ATA GCC ATC CGA GCC AG
NM_007382MCAD 610AAG ACC AAA GCA GAG AAG AAG CAT TGT CCA AAA GCC AAA CC
NM_008212SCHAD 540GGA CCA AAC GGA AGA CAT C GGA CTG GGC TGA AAT AAG GG
NM_013556HPRT176GCG TCG TGA TTA GCG ATG ATG AAC CCT CCC ATC TCC TTC ATG ACA TCT
Carnitine Deficiency & Necrotizing Enterocolitis
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OH) and F4/80 (1:1000) (Abcam Labs. Cambridge, MA). The
staining was done as described earlier . Sections were then
incubated with Alexa Fluor 555–conjugated IgG (Invitrogen,
Carlsbad, CA) secondary antibody (1:1000). Slides were washed in
PBS, incubated with a 1:24,000 dilution of Hoechst stain,
coverslipped, and viewed with an epifluorescence microscope
(Axioplan-2 equipped with the Axiovision Program and an HRM
camera; Carl Zeiss Meditec, Oberkochen, Germany). All im-
munostaining experiments were performed on at least 6 sets of gut
tissue from 1-week-old mice for all enzymes and F4/80 positive
Carnitine Analysis by Tandem Mass Spectrometry
OCTN22/2small intestine mucosal scrapings, spleen, and thymus
were collected and protein concentrations of each sample was
estimated by the Lowry method. The total, free and acyl-carnitine
fractions were determined by tandem mass spectrometry as
described  and results were expressed as carnitine content
per mg of tissue.
Apoptosis Assay in Gut, Spleen and Thymus Tissue
Activity of Caspase-3 was determined in small intestinal
mucosal scrapings using a colorimetric method, CaspACE assay
systemTM, (Promega, Madison, WI) per the manufacturer’s
recommendations. To identify apoptosis in the spleen and thymus,
the ApopTag In Situ Oligo Ligation (ISOL) technique was used
with the T4 DNA ligase kit (Chemicon International, Temecula,
CA) per the manufacturer’s recommendation.
Flow Cytometry for Lymphocyte Subpopulation
Lymphocytes from spleen, thymus and lymph nodes were
obtained by straining the gently crushed intact organ through
a nylon mesh. Erythrocytes were lysed by treatment with an
ammonium chloride solution. Cells were stained on ice. Viable
cells were identified by gating on forward and side scatter, and
CD4+, CD8+, CD24+and CD45-B220+cells were sorted using
magnetic beads coated with individual antibody (MACS cell
sorter, Miltenyi Biotec Inc. Auburn, CA, 95602, USA).
Table 2. Tissue carnitine content of small intestine, spleen and thymus of OCTN2 mice and total lymphocyte count of spleen,
thymus and lymph nodes isolated from each mouse (n=6).
Tissue Carnitine content (nmoles/mg) Total Lymphocyte count (6106/mm3)
Small IntestineSpleen Thymus SpleenLymph node Thymus
9.261.9 11.0462.99.8261.9840.262.2 19.661.919.861.0
2.160.2 0.1260.010.260.1 4.0660.82.4760.9 3.5360.8
Figure 1. Gut histology of 1-week-old wild type (OCTN2+/+) and age-matched homozygous (OCTN22/2) mice (H&E staining, X20).
Images A–C represent photomicrographs of wild type (OCTN2+/+) jejunum, ileum and colon respectively and images D–F represent
photomicrographs of homozygous (OCTN22/2) jejunum, ileum and colon at the same magnification. (Bar represents 50 mm).
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Cytokine Bead Assay
The direct quantification of cytokines was done using a cyto-
metric bead assay (CBA) (BD Biosciences, San Diego, CA) per the
manufacturer’s recommendations. The assay is based on multiple
flow cytometric bead-based immunoassays that measures different
cytokines simultaneously, from a single cell culture supernatant.
We used 3–46105cells per well of total lymphocytes from
OCTN22/2and OCTN2+/+mice. The dye, incorporated in the
beads fluoresces strongly at 650 nm (measured as FL3 signals in
BD FACScan and FACScalibur Flow Cytometers) when excited
with an argon laser. Detection is mediated by the binding of
specific detection antibodies that are directly conjugated with
phycoerythrin (PE), to each of the corresponding capture bead/
Figure 2. Immunohistochemical analysis of macrophage infiltration in 1-week-old wild type (OCTN2+/+) and homozygous (OCTN22/
2) mice ileum sections. Panel A is an ileum section showing F4/80 staining (brown) of villous macrophages from the wild-type mouse and panel C
is a high power magnification of a representative area to help count the number of F4/80 positive cells. Panel B is an ileum section of a homozygous
mouse and panel D is a high power magnification of a representative area. Panel E is a graphic representation of the number of F4/80 positive cells in
each section (Bar represents 100 mm).
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analyte complex populations, thus providing an FL2 fluorescent
signal on the appropriate bead. This signal is proportional to the
concentration of the cytokine in the test matrix. This kit combines
beads with the ability to measure the levels of IL-2, IL-6, MCP-1,
TNF-a and interferon-c from the same sample. Cytokine
Figure 3. Immunohistochemical analysis of expression of fatty acid oxidation enzymes in 1-week-old wild type (OCTN2+/+) mouse
jejunum. Panel A is a negative control image where there was no primary antibody and panels B–F shows immunoreactivity for MCAD, LCAD,
VLCAD, SCHAD, and LKAT, respectively. All five enzymes are expressed in villous epithelial cells and minimal expression in non-epithelial cells of the
villous core. (Bar represents 20 mm).
Figure 4. Immunohistochemical analysis of expression of fatty acid oxidation enzymes in 1-week-old wild type (OCTN2+/+) mouse
ileum. Panel A is a negative control image where there was no primary antibody and panels B–F show immunoreactivity for MCAD, LCAD, VLCAD,
SCHAD, and LKAT, respectively. All five enzymes are expressed mainly in the villous epithelial cells and minimal expression in non-epithelial cells of
the villous core and crypts. (Bar represents 50 mm).
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concentration was measured by using established calibration
curves and dedicated CBA analysis software.
Realtime PCR for Expression of Genes Involved in
Enterocyte Growth, Differentiation, and Migration
We used RT2profiler PCR arrays (SABiosciences, Qiagen,
Frederick, MD) to study differences in gene expression of TGF-
b/BMP pathways (cat No. PAMM035) between small intestinal
mucosal scrapings from 1-week-old OCTN22/2mice and age-
matched wild type controls. RNA was extracted using the Trizol
method from gut tissue from two sets of animals. This array is
housed on a 96-well plate; it provides information on 84 genes
which includes members of the TGF-b superfamily of cytokines
and their receptors: SMAD and SMAD target genes, which
include adhesion, extracellular molecules and transcription factors
involved in downstream cellular processes. Details of this PCR
arrayare available at the
Relative expression levels of each gene were analyzed using
BioRad iQ iCycler Detection System (BioRad Laboratories,
Hercules, CA, USA). All assays were performed per the
manufacturers’ recommendations, each experiment was per-
formed in duplicate, and results represented as the mean of each
Qiagen website (http://www.
All data are presented as means 6 SD and comparisons
between paired samples were made by Student’s‘t’ test with
Bonferroni’s correction where applicable and statistical signifi-
cance was set at p # 0.05. We used statistical software SPSS for
PC version 11.01 for data analysis.
Figure 5. Composite image of semi-quantitative RT-PCR (densitometry) in small intestine mucosal scrapings for MCAD, LCAD,
VLCAD, SCHAD, LCHAD and LKAT (Panel A to F) expression in 1-week-old OCTN2+/+(black bar) and OCTN22/2mouse (white bar)
respectively. Expression of HPRT was used to normalize data from each group. Asterisks (*) represents a statistically significant change in
Figure 6. Caspase 3 activity in 1-week old small intestine
mucosal scrapings from OCTN2+/+(black bar) and OCTN22/2
mice (white bar). Asterisk (*) represents statistically significant
difference in activity.
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The homozygous (OCTN22/2) mice survive for about 4–5
weeks and look much smaller without carnitine supplementation
compared to age-matched wild type (OCTN2+/+) littermates while
they are indistinguishable when 1-week-old. The carnitine content
in small intestine, spleen and thymus of neonatal OCTN22/2
mouse is markedly lower than in the age-matched wild type
1-week-old OCTN22/2Mice Showed Signs of Poor Villous
Growth and Differentiation throughout the GI Tract with
Areas of Lymphocytic and Macrophage Infiltration
By the end of the first week of life the carnitine deficient
OCTN22/2mice develop early signs of stunted villous growth in
comparison to the wild-type mice (Fig. 1) and some areas of the gut
show disruption of the villous architecture with lymphocytic
infiltration and thickening of the muscular mucosa (Fig. 1, panel C
& D). The colonic villi are not as healthy looking and the number
of mucin producing goblet cells is markedly reduced (Fig. 1, panel
E & F). F4/80 staining for macrophages showed a higher number
of macrophages in the villous core of the OCTN22/2ileum
sections (Fig. 2, panel B &D) compared to the age-matched wild-
type controls (Fig. 2 panel A & C). The total number of
macrophages in two respective high power field are quantified in
Fig. 2, panel E.
Five Key Enzymes Involved in Mitochondrial b-oxidation
are Expressed in Enterocytes Lining the Small Intestinal
There was high expression of MCAD, LCAD, VLCAD,
SCHAD and LKAT in the enterocytes lining the villi of jejunum
(Fig. 3) and ileum (Fig. 4) of 1-week-old wild-type (OCTN2+/+)
mice. The enzyme expression (red fluorescence) was mainly
localized to enterocytes over the villous tips with much less
expression in villous core and villous crypts. There was complete
absence of staining in negative control sections (Panel A, Fig. 3&4).
Figure 7. Gross appearance of thymus and spleen. Panel A is an image of OCTN2+/+mouse thymus covering the heart (Arrowhead), and panel
B is an image of OCTN22/2mouse thymus above the heart (Arrowhead). Panel C is an image of age-matched OCTN2+/+(left) and OCTN22/2(right)
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Up-regulation of Genes of 6 Enzymes Involved in b-
oxidation in 1-week-old OCTN22/2Mice
Fig. 5 is a composite image of semi-quantitative RT-PCR
densitometry data for these enzymes. There was a significantly
increased expression of MCAD, LCAD, VLCAD, SCHAD and
LKAT genes in small intestinal mucosal scrapings from carnitine-
deficient animals as compared to age-matched wild type
Carnitine Deficiency in OCTN22/2Mice Leads to
Increased Apoptosis in the GI Tract
Activity of caspase 3 was significantly elevated in small intestinal
mucosal scrapings from OCTN22/2animals compared to 1-
week-old age matched wild-type controls (Fig. 6).
OCTN22/2Mice Develop a Severe Immune Phenotype
Characterized by Atrophy of Lymph Nodes, Thymus and
The wild-type (OCTN2+/+) mouse thymus filled up the whole of
superior mediastinum and covered the heart (Fig. 7A, arrowhead)
whereas the age-matched carnitine-deficient (OCTN22/2) mouse
thymus was small, shriveled and barely visible (Fig. 7B, arrow-
head). Likewise the spleen of OCTN22/2mice was pale and
much smaller in size (Fig. 7C). The total lymphocyte count from
thymus, spleen and lymph nodes was much reduced (Table2).
There was markedly increased lymphocyte apoptosis in the
OCTN22/2mouse spleen and thymus as indicated by brown
staining of lymphocytes with the Oligo Apoptaq assay. There were
several ‘‘punched out’’ areas in the spleen and thymus sections
where a complete loss of lymphocytes had occurred (Fig. 8). Flow
cytometry studies showed that carnitine deficiency led to a change
in relative percentage of lymphocyte surface markers. There was
a relative decrease in CD4+splenocytes in the OCTN22/2mouse
and a marked increase in CD45-B220+thymocytes and lympho-
cytes which indicates exposure to extrinsic environmental antigens
and a T-cell response (Table-3).
OCTN22/2Lymphocytes Produce Increased Amounts of
Pro-inflammatory Cytokines Under Basal and Stimulated
Splenocytes and thymocytes from OCTN22/2mice secreted
significantly increased amounts of IL-2, IL-6, TNF-a, MCP-1 and
IFN-c under basal and anti-CD3 antibody-stimulated conditions
Enterocytes of OCTN22/2Mouse Small Intestine have
a Low Expression of TGF-b/BMP Pathway Genes
Table-5 summarizes the changes in expression of genes involved
in the TGF-b/BMP signaling pathway. We found a similar trend
in results of each of the two experiments and data shown represent
Figure 8. Analysis of apoptosis in thymus and spleen using Oligo-ApopTaq assay (620). Panels A & B are OCTN2+/+and OCTN22/2mouse
spleen sections and panels C & D are OCTN2+/+and OCTN22/2mouse thymus sections respectively. (Bar represents 50 mm).
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mean values of the genes with .2 fold change. There was down-
regulation of most TGF-b/BMP pathway genes and 22 genes
showed a significant change. Genes for TGF-b 1 & 3, its receptor
II, BMP 4 & 6, IGFBP3, integrin b 5 & 7, activin and noggin were
significantly down-regulated in the OCTN22/2mouse gut.
Our previous investigations have demonstrated that carnitine
deficiency leads to severe atrophy of the villous structure
throughout the gut with widespread inflammation, perforation
and abscess formation and signs of peritonitis in the adult
OCTN22/2mouse . In this report, we have explored the
early effects of carnitine deficiency on the developing neonatal gut
to investigate as to what role carnitine deficiency might play at the
molecular level before gross changes are seen in the gut. The
neonatal GI tract requires a constant supply of energy to support
its own growth, maturation, nutrient transport function and
maintenance of epithelial barrier function. This energy is pro-
duced by mitochondrial b-oxidation of long-chain fatty acids in
enterocytes where carnitine plays a crucial role. We have earlier
measured activity of two major fatty acid oxidation enzymes, long-
chain L-3-hydroxyacyl CoA dehydrogenase (LCHAD) and short-
chain L-3-hydroxyacyl CoA dehydrogenase (SCHAD) using adult
OCTN2 mice and found that SCHAD and LCHAD activity in
mouse gut is nearly 4 to 10 fold higher than in liver which is
considered the major organ metabolizing fatty acids . Our
current data are in line with this finding and highlight the value of
carnitine during the neonatal period. Carnitine’s role in the GI
tract has recently been highlighted when several reports linked
mutations in genes encoding carnitine transporters OCTN1
(SLC22A4) and OCTN2 (SLC22A5) with Crohn’s disease (CD)
[23,45]. Though carnitine’s immunosuppressive and therapeutic
properties in gut inflammation have been described in the past
[46,47,48,49,50], this is the first report about its role in
development and differentiation of enterocytes and gut inflamma-
tion during the neonatal period. Carnitine’s role in gut associated
immunity has recently been highlighted where it has been shown
to abrogate gut inflammation and prevents lymphocyte apoptosis
[46,48,51,52]. Carnitine has also been used in adults with gut
inflammation due to CD or ulcerative colitis with beneficial effects
[53,54,55]. Carnitine deficiency in our mouse model causes
a global effect on the immune system as evidenced by severe
atrophy and apoptosis of splenocytes, thymocytes, lymph node
lymphocytes as well as intra-epithelial lymphocytes. This was
preceded by an early pro-inflammatory response in the gut with
macrophage and lymphocytic infiltration. Thus OCTN22/2
mouse spleen and thymus phenotypes are part of the same process.
Intestinal epithelial cells normally undergo apoptosis at a very
high rate and apoptotic cells are quickly replaced by newer cells in
a seamless manner. Any perturbation of this process leads to
breach of the gut epithelial barrier and entry of gut pathogens into
the blood stream. We have demonstrated a major increase in
enterocyte apoptosis in our mouse model of carnitine deficiency
which initiates this process of gut injury. Our earlier studies using
the adult OCTN22/2have shown a higher expression of caspase
1 and 3 in small intestine by Western blot. We also found up-
regulation of gut protective molecules such as phosphorylated
ERK and AKT . Several growth and transcription factors are
required for the transformation of crypt stem cells into mature
enterocytes and secretory cells and ensure integrity of gut mucosal
barrier. These growth factors including the TGF-b/BMP are
involved in various aspects of gut development and differentiation
[56,57,58,59,60]. TGF-b/BMP act through their receptors and
activate a number of intracellular SMAD transcriptional regula-
tors [58,60]. SMAD complexes interact with either co-activators
or co-repressors of transcription to control target gene expression.
Here we have presented data to show that TGF-b/BMP pathway
Table 3. Relative populations of CD4+, CD8+, CD24+and CD45-B220+lymphocytes from spleen, thymus and lymph nodes of wild-
type (OCTN2+/+) and homozygous (OCTN22/2) mice (n=6).
SpleenThymus Lymph node
CD 4CD 8 CD 24 CD45-B220CD 4CD 8 CD 24CD45-B220 CD 4 CD 8 CD 24CD45-B220
17.361.564.267.4 7.261.2 23.462.43.762.3 4.161.3 2.860.6 1.160.1 49.065.8 14.165.9 19.563.9 1.2160.1
26.862.7 55.864.3 10.161.5 25.562.1 7.064.7 6.462.3 3.862.5 40.962.2 50.967.8 14.564.2 14.561.618.261.8
P value0.010.23 0.08 0.3 0.23 0.16 0.45
, ,0.0010.650.82 0.11
Table 4. Basal and anti-CD3 antibody stimulated cytokine production by lymphocytes from of wild-type (OCTN2+/+) and
homozygous (OCTN22/2) mice (n=6).
Basal cytokine production (pg/ml) Anti-CD3 stimulated cytokine production (pg/ml)
IL-6 13.862.360.265.4* 600654.2 880665.4*
MCP-1 0.460.2 1.860.339096245.6 41766466.2
Asterisk (*) represents a statistically significant difference.
Carnitine Deficiency & Necrotizing Enterocolitis
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genes are down-regulated in states of carnitine deficiency and thus
will affect villous growth and differentiation. TGF-b/BMP
pathway genes also regulate cytokine production and mucosal
inflammation  and our studies in the OCTN22/2mouse show
initiation and progression of immune response and increased pro-
inflammatory cytokine production.
Results of our study, though conducted in an animal model, are
relevant to cases of NEC, a disease which predominantly affects
low birth weight preterm neonates since they are born with low
tissue levels of carnitine and may not have received carnitine
supplementation leading to an even lower tissue level of this
conditionally essential nutrient. The heterozygous OCTN2+/2
mouse with lower tissue levels of carnitine develops a clinical
phenotype comparable to the homozygous OCTN22/2mouse in
adult life though its GI pathology has not been reported .
Nutrition plays a central role in causation of NEC as indicated by
marked reduction in its incidence by use of human breast milk.
More than 50% of calories in human breast milk are from fatty
acids indicating that carnitine plays a significant role in the
metabolism of these calories from fatty acids. The problem of low
tissue carnitine levels in the preterm neonate may go unrecognized
by physicians and may thus contribute to gut injury as commonly
seen in NEC. Our animal model of carnitine deficiency is not
a classic model of NEC where NEC is either created by using
caustic agents like dextran sulphate or with LPS instillation in the
stomach followed by subjecting animals to periodic hypoxia, but it
does show the spontaneous changes in molecular events in the
developing enterocytes which ultimately lead to gut injury. Thus
our study supports the notion that carnitine supplementation is
beneficial to preterm neonates who are at high risk of developing
Conceived and designed the experiments: SS PSS VG LI. Performed the
experiments: SS DM PSS VG LI. Analyzed the data: SS DM PSS VG LI.
Contributed reagents/materials/analysis tools: DM PSS VG LI. Wrote the
paper: SS DM PSS VG LI.
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NM_009612 Activin A receptor II
26.7 NM_019919 Latent TGF-b binding protein 1
NM_007554Bone morphogenetic protein 4
22.9 NM_175641 Latent TGF-b binding protein 4
NM_007556 Bone morphogenetic protein 6
NM_007669 Cyclin-dependent kinase inhibitor 1A (P21)
24.1NM_007430 Nuclear receptor subfamily 0,
group B, member 1
NM_009930Procollagen, type III, a1
25.9 NM_008873 Plasminogen activator, urokinase
NM_008343 Insulin-like growth factor BP-3
23.5 NM_008539MAD homolog 1 (Drosophila)
NM_010580 Integrin beta 5
27.2 NM_029438SMAD specific E3 ubiquitin
protein ligase 1
NM_013566Integrin beta 7
22.7NM_009283 Signal transducer and activator
of transcription 1
NM_008416 Jun-B oncogene
25.9 NM_011577Latent TGF-b 1
NM_009371 TGF-b receptor II
23.0NM_009368 Latent TGF-b 3
NM_001013025 TGF-b receptor associated protein 1
24.0NM_009369Latent TGF-b induced
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