Aminopeptidase N Gene Expression and Abundance in Caprine Mammary Gland is Influenced by Circulating Plasma Peptide

Abstract

This study examined the localization and the effect of circulating peptides on the expression of aminopeptidase N (EC 3.4.11.2) in caprine mammary gland. Four lactating goats in mid to late lactation were used in a crossover design and were subjected to 2 dietary treatments. Abomasal infusion of casein hydrolysate was used to increase the concentration of peptide-bound amino acid in the circulation. Samples of mammary gland tissue from each goat were taken by biopsy at the end of each treatment period to measure gene and protein expression of aminopeptidase N in the tissue. There were no measurable effects on feed intake and milk production for any of the treatments. Western blot analysis showed that aminopeptidase N is located on the basolateral side of parenchymal cells and not on the apical membranes. Abomasal infusion of casein hydrolysate caused a marked change in the profile of arterial blood free amino acids and peptide-bound amino acids smaller than 1500 Da. Abundance of aminopeptidase N mRNA and protein increased by 51 and 58%, respectively, in casein hydrolysate-infused goats compared with the control treatment. It was concluded that aminopeptidase N is one candidate actively involved in the mammary gland to support protein synthesis and milk production. In accordance with the nutritional conditions in the current experiment, it is suggested that aminopeptidase N expression is partly controlled by the metabolic requirements of the gland and postabsorptive forms of amino acids in the circulation.

Full text

J. Dairy Sci. 88:2055–2064
© American Dairy Science Association, 2005.
Aminopeptidase N Gene Expression and Abundance in Caprine
Mammary Gland is Influenced by Circulating Plasma Peptide
S. J. Mabjeesh,
1
O. Gal-Garber,
1
J. Milgram,
2
Y. Feuermann,
1
M. Cohen-Zinder,
1
and A. Shamay
3
1
Department of Animal Sciences, Faculty of Agricultural, Food and Environmental Quality Sciences, and
2
The Koret School of Veterinary Medicine, The Hebrew University of Jerusalem, PO Box 12, Rehovot, Israel
3
Agricultural Research Organization, Volcani Center, Institute of Animal Science, Bet Dagan, Israel
ABSTRACT
This study examined the localization and the effect
of circulating peptides on the expression of aminopepti-
dase N (EC 3.4.11.2) in caprine mammary gland. Four
lactating goats in mid to late lactation were used in a
crossover design and were subjected to 2 dietary treat-
ments. Abomasal infusion of casein hydrolysate was
used to increase the concentration of peptide-bound
amino acid in the circulation. Samples of mammary
gland tissue from each goat were taken by biopsy at
the end of each treatment period to measure gene and
protein expression of aminopeptidase N in the tissue.
There were no measurable effects on feed intake and
milk production for any of the treatments. Western blot
analysis showed that aminopeptidase N is located on
the basolateral side of parenchymal cells and not on
the apical membranes. Abomasal infusion of casein hy-
drolysate caused a marked change in the profile of arte-
rial blood free amino acids and peptide-bound amino
acids smaller than 1500 Da. Abundance of aminopepti-
dase N mRNA and protein increased by 51 and 58%,
respectively, in casein hydrolysate-infused goats com-
pared with the control treatment. It was concluded that
aminopeptidase N is one candidate actively involved in
the mammary gland to support protein synthesis and
milk production. In accordance with the nutritional con-
ditions in the current experiment, it is suggested that
aminopeptidase N expression is partly controlled by the
metabolic requirements of the gland and postabsorptive
forms of amino acids in the circulation.
(Key words: mammary gland, aminopeptidase N,
peptide)
Abbreviation key: AP = alkaline phosphatase, APN =
aminopeptidase N, CAS20 and CAS40 = casein hydrol-
ysate infusions providing an additional 20 and 40%
MP, respectively, CAS30 = casein hydrolysate infusion
Received December 7, 2004.
Accepted March 10, 2005.
Corresponding author: S. J. Mabjeesh; e-mail: Mabjeesh@agri.
huji.ac.il.
2055
replacing 30% of MPI, FAA = free amino acid, γ-GT = γ-
glutamyl transpeptidase, MP = metabolizable protein,
MPI = metabolizable protein intake, PBAA = peptide-
bound AA, PMV = plasma membrane vesicles.
INTRODUCTION
Milk from ruminants provides more than 30% of ani-
mal protein in the human diet worldwide (Taylor and
Field, 1998). Researchers and producers are constantly
searching for methods to further increase yield per ani-
mal to improve production efficiency. For example, only
35% (on an additive basis) of AA was converted to milk
protein when cows were supplied with AA to compen-
sate for N deficiency caused by dietary management
(Bequette et al., 1998). The apparent low efficiency of
converting AA to milk protein in the udder could be
related to the fact that the balance measurements
across the mammary gland are done on the basis of net
uptake of free AA from plasma without considering the
contribution of circulating peptide-bound AA (PBAA).
Although, the actual contributions of PBAA to net pro-
tein metabolism in the udder are small, it is important
to consider their metabolic impact (Bequette et al.,
1998).
Transport of PBAA is an important physiological pro-
cess that occurs in tissues of animals (Matthews, 1991).
Peptide-bound AA may constitute a portion of total AA
absorption in ruminants (McCormick and Webb, 1982;
Danilson et al., 1987) and peptide transporters are pres-
ent in tissues of sheep, cows, pigs, and chickens (Mat-
thews et al., 1996a,b; Pan et al., 1997, 2001; Chen et
al., 1999). Northern blot analysis conducted with tis-
sues from sheep and lactating Holstein cows showed the
presence of a 2.8-kb mRNA transcript for the peptide
transporter, PepT1, in the omasum, rumen, duodenum,
jejunum, and ileum (Chen et al., 1999). No hybridiza-
tion was observed with mRNA from the abomasum,
cecum, colon, liver, kidney, and semitendinosus and
longissimus muscles of either species or from the mam-
mary tissue from the cows.
Individual tissues including the mammary gland of
ruminants may be able to use AA residues of PBAA for

MABJEESH ET AL.2056
protein synthesis. Cultured bovine mammary epithelial
cells and tissue explants from lactating CD-1 mice used
methionine from methionine-containing dipeptides to
support protein accretion and synthesis of secreted pro-
teins (Pan et al., 1996; Wang et al., 1996). Results from
an in vivo experiment with lactating dairy goats indi-
cate that mammary glands use PBAA from the circula-
tion for protein synthesis and secretion (Backwell et
al., 1996). Similar results were reported for dairy cows
receiving different dietary treatments (Tagari et al.,
2004). However, in the absence of peptide transporter
in the mammary gland, the mechanism(s) of action may
be external PBAA hydrolysis and absorption of the lib-
erated AA in the free form.
Recently, it was shown that rodent mammary tissue
expresses a variety of dipeptidases on the basolateral
surface of the epithelial cells that are capable of hy-
drolyzing peptides extracellularly (Shennan et al.,
1998, 1999). Previously, we showed that aminopepti-
dase N (APN; EC 3.4.11.2) is expressed in the mam-
mary gland of goats and cows (Mabjeesh et al., 2001).
Aminopeptidase N enzyme plays an important role in
protein digestion and absorption in the small intestine;
its activity in the plasma membrane of the mammary
epithelium may explain how PBAA from the circulation
are internalized by the mammary gland (Mabjeesh et
al., 2001).
The purpose of the current study was to investigate
the location of APN in the mammary gland and whether
the concentration of circulating peptides affects and
controls the expression of APN in the mammary gland
in vivo. For this experiment, goats in mid to late lacta-
tion were used. The abomasal infusion of casein hydrol-
ysate was used to increase the concentration of PBAA
in the circulation. Some of the results of this experiment
have been published in preliminary form (Mabjeesh et
al., 2001).
MATERIALS AND METHODS
Animals, Feeding, and Treatments
All surgical and experimental procedures were ap-
proved by the Animal Care and Ethics Committee of
the Hebrew University of Jerusalem. Two experiments
were conducted. The first was preliminary and was con-
ducted on 2 multiparous Israeli Saanen dairy goats in
late lactation (180 ± 30 DIM and 879 ± 97 g/d of milk).
Goats were surgically prepared with abomasal cannu-
las to allow infusion and a raised carotid artery to allow
arterial blood sampling. Goat handling, management,
and the abomasal infusion protocol were similar to the
second experiment (see details below). Goats were as-
signed to 3 experimental periods that each lasted 14 d.
Abomasal infusates were water or water plus casein at
Journal of Dairy Science Vol. 88, No. 6, 2005
2 levels of metabolizable protein intake (MPI;20and
40%; basal MPI = 116.3 ± 7.2 g/d) above that available
to the goats from the diet, e.g., infusate increased the
actual supply of metabolizable protein (MP)by20and
40% of the daily intake of MP. The order of treatments
that each goat received was control, casein hydrolysate
with 20% increased MP (CAS20), and casein hydroly-
sate with 40% increased MP (CAS40). Milk production
and feed intake were monitored over the last 7 d of
each period.
The second experiment was conducted on primipa-
rous Israeli Saanen goats (n = 4; BW = 55 ± 6 kg) in
mid to late lactation (160 ± 25 DIM). Goats were used
in a crossover design. In period 1, goats were randomly
assigned (2 per treatment) to 2 dietary treatments, and
one udder half was randomly selected for monitoring
and sampling. During period 2, dietary treatments were
switched and the contralateral udder half was moni-
tored and sampled. Goats were surgically prepared with
abomasal cannulas. Two goats were prepared by raised
carotid arrangement as described above.
Goats were placed in metabolism crates and allowed
at least 10 d of adaptation to frequent feeding of diets
by automatic feeders (12 equal portions daily at 2-h
intervals) and the daily routines of machine and hand-
milking (0700 and 1900 h). Milk weights were recorded
at each milking. The diet was formulated to meet me-
tabolizable energy and protein requirements for main-
tenance and milk production (AFRC, 1992, 1993) and
contained 40% chopped vetch-clover hay, 60% concen-
trate feeds, and relevant vitamin and mineral mixes
(Table 1). The concentrates contained commercial pel-
lets (1474, Matmor Ltd., Ashdod, Israel), barley, and
corn whole grains. Daily feed refusals were collected
and weighed, and feed intake was adjusted to allow
10% refusals.
Treatments in the preliminary study were: 1) control,
abomasal infusion of water (1000 g/d), 2) abomasal infu-
sion of casein hydrolysate (Sigma no. A-2427; Sigma-
Aldrich, Ltd., Rehovot, Israel) CAS20, and 3) abomasal
infusion of casein hydrolysate CAS40 dissolved (emulsi-
fied) in 1000 g/d of H
2
O. Infusion was conducted by
peristaltic pump (Minipuls 2, Gilson, Paris, France).
The quantity of casein hydrolysate infused was fixed
to deliver an added 20 or 40% of the MPI and was
adjusted daily based on feed intake. Small intestine
digestibility of casein was assumed 100% in accordance
with our previous in vivo findings with sheep (Mabjeesh
et al., 2003), thus all CP of casein (81.7 g/100 g of raw
material) was considered MP.
Treatments in the main study were: 1) control–abo-
masal infusion of H
2
O (1000 g/d), 2) abomasal infusion
of casein hydrolysate (Sigma no. A-2427; Sigma-Ald-
rich) dissolved (emulsified) in 1000 g/d of H
2
O. Infusion

AMINOPEPTIDASE N IN THE MAMMARY GLAND 2057
Table 1. Composition of diet and chemical analysis given to goats.
%of %DM
Ingredient DM of diet
Grain pellets 1474
1
30
Soybean meal 15.3
Rapeseed meal 10.0
High fat product 4.52
NPN (mixture)
2
0.43
Poultry oil 0.5
Barley grains 12.41
Corn grain 10.0
Wheat grain 20.0
Sunflower meal 6.10
Gluten feed 10.0
Soybean hulls 8.0
Salt/calcium 2.14
Sodium sulfate 0.007
Vitamin E, 50% 0.014
Vitamins and minerals
3
0.076
Whole barley grain 15
Whole corn grain 15
Chopped vetch-clover hay 40
Chemical analysis of diet
CP 18.2
Metabolic protein 14.5
OM 94.0
RUP 5.18
Metabolic energy, Mcal/ kg 2.61
1
Grain mixture 1474 (Matmor Ltd., Ashdod, Israel).
2
Contained 80% urea and 20% ammonium sulfate.
3
Contained 20,000,000 IU of vitamin A/kg, 2,000,000 IU of vitamin
D/kg, 15,000 mg vitamin E/kg, 6000 mg/kg of Mn, 6000 mg/kg of Zn,
2000 mg/kg of Fe, 1500 mg/kg of Cu, 120 mg/kg of I, 50 mg/kg of Se,
and 20 mg/kg of Co.
was conducted by peristaltic pump (Gilson). The quan-
tity of casein hydrolysate infused (CAS30) was fixed to
replace (not extra, as in the pilot study) 30% of the
MPI and was corrected daily according to refusals. To
calculate the daily MPI and the casein infusate, a
spreadsheet was created with the diet ingredients (pel-
lets, corn and barely grains and hay) and their corres-
ponding chemical composition of CP, RUP, RDP, and
rumen-degradable OM. These parameters were mea-
sured in situ, and MP was calculated in accordance to
AFRC model (1992) and the model suggested previously
(Arieli et al., 1989). The pellets were considered the
major supplement of MP and, according to daily cor-
rected feed intake, the amount was changed in order not
to exceed the total calculated MPI. To prevent severe
alterations in microbial CP flow, rumen-degradable OM
was calculated for the diet and kept at the semioptimal
concentration by manipulating the amount of barley
and corn grain in the diet. A ratio of 5:1 of rumen-
degradable OM:RDP was considered ideal for keeping
optimal microbial CP synthesis in the rumen (Arieli et
al., 1989).
Journal of Dairy Science Vol. 88, No. 6, 2005
Sample Collection and Analysis
In the preliminary study, on the last day of each
experimental period, goats were machine milked at
0700 h. Then, polyvinyl catheters were implanted into
the carotid artery to allow blood withdrawal. Four blood
samples (10 mL) were withdrawn from the artery every
hour (n = 4). Plasma was immediately separated by
centrifugation at 3000 × g for 15 min at 4°C and was
stored at −20°C for further analysis.
On the last day of each experimental period in the
main study, goats were machine milked at 0700 h, and
mammary tissue samples (1.5 to 2.0 g) were taken by
biopsy. Four arterial blood samples were taken from 2
goats, as described earlier, before the biopsy was per-
formed. For the biopsy procedure, goats were sedated
with xylazine (Rompum, Teva Medical, Ltd., Ashdod,
Israel) and local anesthesia (lidocaine, 2%; Teva Medi-
cal) was introduced to the area (at the midupper part
of the udder) of the udder half that was being monitored.
Tissue samples were immediately divided into 2 por-
tions, one of which was kept in RNAlater storage/stabi-
lization solution (Ambion, Inc., Austin, TX) at 4°C and
the other one was frozen with liquid N and stored at
−80°C until analysis.
Plasma AA and PBAA Analysis
Arterial plasma was prepared for free amino acid
(FAA) and PBAA analysis by the method of Backwell
et al. (1997). Plasma proteins were precipitated by com-
bining plasma with 1 M perchloric acid (1:1 vol/vol)
that contained norleucine (25.6 mg/L) as an external
standard. The combination was mixed thoroughly.
Samples were centrifuged at 1500 × g at 4°C for 15 min,
and the supernatant was re-centrifuged under the same
conditions to remove any residual protein. The superna-
tant was then neutralized (pH 7 to 8) by adding 2 M
K
2
CO
3
and allowed to stand for 2 h at 4°C before the
precipitated perchlorate salt was removed by centrifu-
gation as described above. The supernatant was applied
to a Sephadex G-15 column (volume 5 mL; Pharmacia
Biotech AB, Uppsala, Sweden) equilibrated in 0.2 M
ammonium bicarbonate, pH 8.0, and eluted with the
same buffer at a flow rate of 0.3 mL/min on an A
¨
KTA-
prime fast performance liquid chromatography system
(Amersham Pharmacia Biotech AB). The column void
volume (2 column volumes, approximately 10 mL),
which contained residual soluble protein or peptides of
molecular weight > 1500 Da, was discarded and, there-
after, fractions that contained peptides of MW < 1500
Da were collected, pooled, and lyophilized. Dried sam-
ples were stored until analyzed for FAA and PBAA
by HPLC using the Pico-Tag method (Waters Corp.,
Milford, MA) (Bidlingmeyer et al., 1984). The PBAA

MABJEESH ET AL.2058
content of samples was calculated as the difference be-
tween the corrected AA content of hydrolyzed samples
and the FAA content of the same sample before hy-
drolysis.
RNA Preparation
Total RNA was isolated from the mammary gland
tissues kept in RNAlater using TRI reagent (1 mL/100
mg of tissue) according to the manufacturer’s protocol
(MRC Molecular Research Center, Inc., Cincinnati,
OH).
Reverse Transcription-Polymerase Chain Reaction
The following primers were used (based on conserved
regions of the published genes): forward: 5′-CTGGGG
ACTGGTGACCTACCGGG-3′; reverse: 5′-CGCTGGAC
CCTCGAGATGGGCTT-3′ in the reverse transcription-
PCR reaction. Total RNA was amplified using PCR
Sprint equipment (Hybaid, Ltd., London, UK) utilizing
the Promega Access RT-PCR system according to the
manufacturer’s protocol (Promega Corporation, Madi-
son, WI). One microgram of total RNA was added to 1×
AMV/Tfl 5× reaction buffer, 0.2 mM dNTP mix (10 mM
each dNTP), 1 μM upstream and downstream primers,
1mM MgSO
4
, 0.1 U/μL AMV reverse transcription (5
U/μL), 0.01 U/μL Tfl DNA polymerase (5 U/μL), and
diethyl pyrocarbonate-treated water in a total volume
of 50 μL. The reaction tubes were incubated for 1 cycle
at 48°C for 45 min (for reverse transcription), 1 cycle
at 94°C for 2 min (AMV RT inactivation and RNA/
cDNA/primer denaturation), 35 cycles at 94°C for 30 s
(denaturation), 60°C for 1 min (annealing), 68°C for 2
min (extension), and 1 cycle at 68°C for 7 min (final ex-
tension).
The reverse transcription-PCR products were exam-
ined on a 1.5% agarose gel, visualized by staining with
ethidium bromide, excised from the gel, and purified
with a gel extraction column (Wizard PCR Preps DNA
purification system, Promega). The mammary gland
aminopeptidase cDNA fragment was subjected to auto-
mated sequencing using an Applied Biosystem 373A
DNA sequencer (Applied Biosystems). Nucleic acid se-
quences were analyzed using the GCG Wisconsin suite
of programs (GCG, San Diego, CA) (Devereux et al.,
1984). The homology between goat and other aminopep-
tidase sequences was calculated using DNAMan ver-
sion 4 (Lynnon Biosoft 1994—1997, Lynnon Bioinfor-
matic Solution, Quebec, Canada).
Northern Blot
For Northern blot analysis, 30 μg of total RNA was
denatured and separated by electrophoresis on 1.5%
Journal of Dairy Science Vol. 88, No. 6, 2005
agarose/1.1 M formaldehyde gel. After electrophoresis,
RNA was transferred overnight by capillary transfer
to a nylon filter (Hybond-N; Amersham Pharmacia Bio-
tech) and then fixed on the filter by UV at 340 nm for
2 min.
Hybridization
Two probes were used for hybridization: 1) The iso-
lated cDNA fragment of goat mammary gland amino-
peptidase, and 18S cDNA (Ambion, Inc.) to normalize
variations in the total RNA loading. The probes were
labeled with
32
P-dCTP by the random prime labeling
method (Biological Industries, Kibbutz Beit Haemek,
Israel). Prehybridization was done at 42°C for 4 h, hy-
bridization was at 42°C overnight, and a high-strin-
gency wash (0.1× saline sodium citrate/ 0.1% SDS at
60°C) was conducted according to the procedures recom-
mended by Amersham for Hybond N membranes (Am-
ersham Pharmacia Biotech). Blots were exposed for 24
hat−70°C to Kodak XAR 5 film in the presence of an
intensifying screen.
Preparation of Plasma Membrane Vesicles
Plasma membrane vesicles (PMV) from the basal
side of the parenchymal cells taken from frozen caprine
mammary tissue were prepared using MgCl
2
precipita-
tion and sequential centrifugation as descried by Vayro
et al. (1991). Activity of membrane markers such as
alkaline phosphatase (AP; EC 3.1.41) and γ-glutamyl
transpeptidase (γ-GT; EC 2.3.2.2) in the PMV and ho-
mogenates were measured to validate the quality of the
preparation. The enzymatic activity of AP and γ-GT
were measured according to Sigma Diagnostics’ kits
(cat. no. 221 and 419, respectively, Sigma-Aldrich, Inc.).
p-Nitrophenyl phosphate and
L
-Leu p-nitroanilide were
used as substrates in the reaction to measure the activ-
ity of AP and γ-GT. The accumulation over time of p-
nitrophenyl and p-nitroanilide in the reaction medium
was measured spectrophotometrically at 410 and 405
nm, respectively. The reactions were performed at 37°C
in cuvette cells in the spectrophotometer (UVIKON 810,
Kontrom Analytical, Bunnik, Switzerland) over 7 min
and the absorbance was recorded each 1 min. The en-
richment activity of AP and γ-GT was 11.57 and 8.85
times the original homogenate, respectively. Mem-
branes from the apical side were prepared from bovine
milk as described previously (Shennan, 1992). Bovine
milk was used to isolated apical membrane because fat
globules contain only membranes from the epithelial
tissue (secretory cells). Fat globules from caprine milk
contain a larger portion (up to intact cells) of secretory
cell membrane, which makes it difficult to isolate pure

AMINOPEPTIDASE N IN THE MAMMARY GLAND 2059
apical membrane (Shennan, 1992). The final protein
concentration in PMV was 9 to 15 g/L. Aliquots of 50
to 100 μL of PMV were frozen in liquid nitrogen and
stored at −80°C until use.
Western Blot Analysis
The PMV were solubilized in a loading buffer con-
sisting of 1% Triton X-100, 50 mM HEPES, 20% glyc-
erol, and 5% β-mercaptoethanol, and then heated at
100°C for 2 min.
Samples (15 μg of protein) were separated by SDS-
PAGE on 10% gels under reducing conditions (Laemmli,
1970). Following electrophoresis, proteins were trans-
ferred to nitrocellulose membranes (Schleicher and
Schuell, Dassel, Germany). After blocking with TBS-
Tween [50 mM Tris-HCl, pH 7.5, 150 mM NaCl, and
0.5% (vol/vol) Tween 20] containing 3% BSA, mem-
branes were incubated overnight at 4°C with antigoat
polyclonal CD 13 (APN) antibody (1:100; Santa Cruz
Biotechnology, Inc., Santa Cruz, CA). Membranes were
then washed with TBS-Tween and visualized using en-
hanced chemiluminescence by incubating for 1 h with
horseradish peroxidase-conjugated donkey secondary
antigoat IgG (1:15,000; Jackson Immuno Research Lab-
oratories, Inc., West Grove, PA) at 22°C. A mouse mono-
clonal antiAPN (CD 13) IgG
2a
raised against LPS-
treated human U937 cells (1:100; Calbiochem Biochem-
ical and Immunochemical, Darmstadt, Germany) was
also used as above for APN detection in the PMV.
Statistical Analyses
Concentrations of AA were measured on each plasma
sample. The average (n = 4 for each goat) was calculated
and used in the statistical model as follows. Data, in-
cluding gene and protein expression (each was per-
formed in triplicate), were analyzed using ANOVA to
compare treatment effects by the GLM procedure of
SAS (SAS Institute, 1985) in a crossover design. The
linear model included the effect of treatment, period,
goat (random effect), and the residual error term.
Means were separated using Students’ t-test and differ-
ences were considered significant when P < 0.05 unless
stated otherwise. Data are presented as least square
means ± SEM.
RESULTS
Preliminary Experiment
Results of the preliminary experiment are presented
in Figures 1 and 2. All variables measured were in-
creased in a dose-response manner (based on regression
analysis which revealed R
2
= 0.94 to 0.99; P < 0.021 to
Journal of Dairy Science Vol. 88, No. 6, 2005
Figure 1. Concentration of free AA (A) and peptide-bound AA (B)
in arterial plasma of dairy goats (n = 2) abomasally infused daily
with 1000 g of water (CON), casein hydrolysate solution equaling
20% of metabolizable protein intake (CAS20), or casein hydrolysate
solution equaling 40% of metabolizable protein intake (CAS40). Re-
sults are least square means ± SE.
0.001) with the increased casein hydrolysate infusion.
Feed intake was increased (P < 0.01) from 794 g/d up
to 1334 and 1814 g/d when goats were infused with
Figure 2. Dry matter intake and milk production (g/d) of dairy
goats (n = 2) abomasally infused daily with 1000 g water (CON),
casein hydrolysate solution equaling 20% of metabolizable protein
intake (CAS20), or casein hydrolysate solution equaling 40% of metab-
olizable protein intake (CAS40). Results are least square means ± SE.

MABJEESH ET AL.2060
Figure 3. Western blot analysis of aminopeptidase N (APN) with
monoclonal antibody raised against CD13 from human U937 cells.
Lane identification: AM = apical plasma membrane vesicles, BM =
basal plasma membrane vesicles, and CSI = positive control from
brush border membrane vesicles from chicken small intestine, fasted
and fed, respectively.
CAS20 and CAS40, respectively. Milk yield was in-
creased by 68% when goats received CAS20 and by
129% when goats received CAS40, compared with the
control treatment. The essential FAA and PBAA con-
centrations in arterial plasma gave apparent evidence
that the casein hydrolysate was absorbed from the
small intestine and some AA were transported in the
form of peptides. With the exception of Met PBAA, con-
centrations in arterial plasma were increased by 49%
(P < 0.001 to 0.0001) and 150% with the infusion of
CAS20 and CAS40, respectively. Overall, total essential
AA concentrations including PBAA were apparently in-
creased in arterial plasma by 70 and 190% when CAS20
and CAS40 were infused, respectively.
Main Study–Goat Performance
All goats completed the experiment and remained
healthy. Goats fully recovered after each biopsy within
5 to 7 d, and no signs of milk reduction were noticed
after the surgical procedure. Goats on both dietary
treatments produced similar amounts of milk (1000 ±
84 g/d) and consumed 1311 ± 113 g of DM daily. Milk
protein concentration was similar for both treatments
and averaged 38.1 ± 0.14 g/L).
APN Protein Abundance in Mammary Gland
on the Basal Side of the Parenchymal Cells
To detect APN protein abundance, Western blot anal-
ysis was performed on membranes prepared from mam-
mary tissue (goat and cow), milk, and PMV from intesti-
nal brush border of chicken and rat (Figures 3 and 4).
Monoclonal and polyclonal anti-CD 13 antibodies were
used to detect the APN protein in the different tissues
and to ensure the specificity of the polyclonal antibody
to APN.
In all tissues examined, both antibodies detected a
band at approximately 140 kDa. The APN protein was
not detected in PMV prepared from milk. The PMV
preparations from milk are believed to be from the api-
cal side of the epithelial cells in the productive tissue
of the mammary gland (Shennan, 1992).
Journal of Dairy Science Vol. 88, No. 6, 2005
Figure 4. A. Western blot analysis of aminopeptidase N (APN).
Lane identification: RSI = rat small intestine, Gt = plasma membrane
vesicles of lactating mammary goat, Cow = plasma membrane vesicles
of lactating mammary cow, and L = protein ladder. This blot was
performed with goat polyclonal antibody raised against human APN.
B. The inset blot was conducted with monoclonal antibody raised
against human U937 cells. Lane identification: RSI = positive control
(small intestine brush border membrane vesicles of chicks), and lane
Gt = plasma membrane vesicles from lactating goat.
In accordance with these results (Figure 3 and 4),
the polyclonal anti-CD 13 was used for Western blot
analysis in tissues taken by biopsy from goats subjected
to the different treatments.
Effect of Casein Hydrolysate Infusion on Arterial
Plasma FAA and PBAA, mRNA, and Protein
Abundance of APN in Caprine Mammary Gland
Presented in Figure 5 are the FAA and PBAA concen-
trations in arterial plasma of goats (n = 2) receiving
similar MPI in 2 different forms (casein infusion vs. MP
from diet). Results confirm the success of the infusion
treatments. Concentrations of arterial FAA in both
treatments were not significantly different for AA mea-
sured. However, PBAA concentrations were greater (P
< 0.01 to 0.001) for most AA in the casein-infused goats
compared with control goats. It was expected in this
experiment, that when infusate replaced rather than
added MP, that the overall MP would be similar for both
treatments (control and CAS30); however, the plasma
profile of AA shifted toward higher (P < 0.05) PBAA in
the circulation (Figure 5).
The mammary gland aminopeptidase cDNA frag-
ment was analyzed, and the nucleic acid sequence was
published in GenBank (AJ304432). The homology be-
tween goat and other aminopeptidase sequences was
calculated and averaged 76 to 84% for chicken and hu-
man intestine, respectively.

AMINOPEPTIDASE N IN THE MAMMARY GLAND 2061
Figure 5. Concentration of free AA (A) and peptide-bound AA (B) in arterial plasma of dairy goats (n = 2; main study) abomasally infused
daily with 1000 g of water (䊐) or casein hydrolysate solution that replaced 30% of metabolizable protein intake (䊏). Results are least square
means ± SE.
Aminopeptidase N mRNA abundance detected by
Northern blot analysis (Figure 6) was increased by 51%
(P < 0.05) in goats undergoing casein hydrolysate infu-
sion compared with control animals. The APN protein
appearance on PMV prepared from mammary tissues
of goats changed in a manner similar to mRNA expres-
sion. Protein expression of APN was increased by 58%
(P < 0.02) in the casein hydrolysate-treated goats com-
pared with controls (Figure 7).
DISCUSSION
The primary aim of the current study was to investi-
gate whether PBAA concentration in the circulation
Journal of Dairy Science Vol. 88, No. 6, 2005
affects expression of APN in the mammary gland. We
proposed that APN could be one of the mechanisms
involved in acquiring substrates to be used for milk
protein synthesis and secretion. Hence, its expression
should be sensitive to any metabolic challenge at the
substrate level. Based on published results (Shennan
et al., 1999, 1998), APN might be one of the peptidases
involved in providing AA for protein synthesis in the
gland. Moreover, the mechanisms of how the activity
of this enzyme might be affected by circulating peptides
is one step toward understanding the metabolic path-
ways by which AA and PBAA are used by the lactating
mammary gland of ruminants. Hence, casein hydroly-

MABJEESH ET AL.2062
Figure 6. Northern blot analysis of aminopeptidase N (APN) ex-
pression in mammary gland tissue taken from goats (n = 4) infused
with 1000 g/d of water (䊐) or with casein hydrolysate solution that
replaced 30% of metabolizable protein intake in 1000 g/d of water
(CAS30; 䊏). Expression of APN in the CAS30 treatment was in-
creased by 51% (P < 0.05; SEM = 4.72;n=4)compared with the
control treatment. Each blot was conducted in triplicates. Results
are least square means ± SE.
sate infusion into the stomach was used to imitate the
effect of dietary challenges that might cause an increase
in peptide flow and absorption and portal appearance
of PBAA (Delgado-Elorduy et al., 2002).
Figure 7. Western blot analysis of APN expression in mammary
gland tissue taken from goats (n = 4) infused with 1000 g/d of water
(䊐) or with casein hydrolysate solution that replaced 30% of metabo-
lizable protein intake in 1000 g/d of water (䊏). Abundance of APN
in casein hydrolysate-infused goats was increased by 58% (P < 0.02;
SEM = 16.0; n = 4) compared with the control treatment. Each blot
was performed in triplicate. Results are least square means ± SE.
Journal of Dairy Science Vol. 88, No. 6, 2005
Indeed, the results of the preliminary and the main
experiments confirmed that at least a portion of MP
was absorbed from the small intestine in the form of
peptides and caused an increased PBAA concentration
in arterial blood. This is in agreement with previously
published data from dairy cows that used the same
approach (Choung and Chamberlain, 1995). Variable
contributions of PBAA to the total concentration of AA
have been reported. For example, Seal and Parker
(1996) reported 26 to 28%, Koeln et al. (1993), Remond
et al. (2000), and Tagari et al. (2004) reported 64, 38,
and 21%, respectively, of PBAA concentrations as a
percentage of total AA concentration. In the prelimi-
nary study, the contribution of essential PBAA in arte-
rial plasma averaged 29% of total AA concentrations
and was similar for all treatments. In the main study,
PBAA contribution ranged from 10% in the control
treatment to 40% in the casein infusion.
Tissue use and absorption of intact peptides is well
recognized across different nutritional states (Adibi
1987; Hubl et al., 1989). The concept of PBAA contribut-
ing to mammary gland metabolism and protein synthe-
sis and secretion has been observed in different studies.
Published data from in vivo studies indicated that the
caprine lactating mammary gland could use many es-
sential AA in the form of PBAA for milk protein synthe-
sis (Backwell et al., 1996; Bequette et al., 1999). The
bovine mammary gland also extracts essential AA from
the circulation in the form of peptides (Tagari et al.,
2004). For example, Met (48 to 71%) and Lys (37 to
60%) are extracted by the mammary gland as PBAA
depending on the dietary treatment. It was suggested
that corn processing might cause greater microbial pro-
tein synthesis in the rumen and thus increase MP flow
to the small intestine, which affects the contribution of
PBAA to the AA flux of portal-drained viscera as well
as to the mammary gland (Tagari et al., 2004).
In vivo studies demonstrated, by kinetic methods
with labeled AA using the precursor-product tracer
technique, that the mammary gland is able to use pep-
tides as a source of AA for protein synthesis under
different dietary conditions such as a shortage of certain
AA or different physiological states (Bequette et al.,
2000; Mabjeesh et al., 2000). The mechanism by which
PBAA uptake occurs is not yet clear. For example,
mRNA for peptide transport systems was not detected
in lactating mammary gland from cows (Chen et al.,
1999). Another route for uptake was suggested, how-
ever, such as peptidase proteins that are embedded in
the basolateral membrane of the parenchymal cells in
the gland. Shennan et al. (1999, 1998) suggested that
transport of intact dipeptides by perfused rat mammary
gland was very low even under conditions designed to
maximize uptake. However, it was shown that the

AMINOPEPTIDASE N IN THE MAMMARY GLAND 2063
mammary gland has a large capacity to hydrolyze di-
peptides on the basolateral side; the contribution of
FAA influx by the lactating mammary gland is quanti-
tatively more significant than uptake of intact peptides
(Shennan et al., 1998).
In the current study, we replaced 30% of the MPI by
a casein hydrolysate infusion (CAS30) to ensure isoni-
trogenous MP supply and to satisfy lactation require-
ments. It was hypothesized that increasing circulating
peptides with a constant MP supply would cause a di-
rect effect on the activity and expression of the mecha-
nism responsible (e.g., APN) for PBAA uptake into the
mammary gland, with minor effects on the level of milk
protein secretion.
It was also important to show that APN is exclusively
expressed on the basal side of the gland (Figure 3 and
4) and not at the apical membrane. This is because
APN is located on intralobular and interlobular fibro-
blasts and on the apical surface of epithelial cells, at
least in human nonlactating breast tissue (Atherton et
al., 1992, 1994). Nevertheless, in the current study,
the basolateral location of APN supports the finding of
Shennan et al. (1998, 1999) that peptides are hy-
drolyzed extracellularly on the blood-facing membrane,
and then FAA are taken up by the gland via high-
affinity transporters. Recently, it was shown that the
enzyme γ-glutamyl transpeptidase is expressed in ovine
mammary gland tissue and is located on the basolateral
side of the productive cells (Johnston et al., 2004). More-
over, it was affected by physiological state, such as preg-
nancy or lactation. It is noteworthy that plasma mem-
branes prepared in the current study may include por-
tions of other supportive tissues in the gland such as
connective tissue.
It appears that APN is influenced by peptide concen-
trations in arterial blood. Northern and Western blot
analysis of mRNA and PMV protein extracted from
mammary tissues showed that increasing the relative
portion of PBAA concentration in the circulation of lac-
tating goats caused a 51 and 58% increase in the gene
expression and protein, respectively. This finding may
be explained as a direct effect of the increase in arterial
peptide concentration, albeit without changing the
overall supply of AA that satisfies milk requirements.
The parallel increase in gene and protein expression
observed in goats subjected to casein hydrolysate infu-
sion supports the concept that mRNA was translated
to protein to support the demand of the tissue for AA
supply for protein synthesis. This might indicate that
the mammary tissue senses the lack of FAA, and metab-
olite signals, which might be extracellular (e.g., PBAA
and FAA) or intracellular to regulate the APN expres-
sion and activity. Similarly, intestinal peptidase activ-
ity is regulated by a mechanism that involves precur-
Journal of Dairy Science Vol. 88, No. 6, 2005
sors and products of peptide hydrolysis in humans
(Sanderink et al., 1988; Kushak and Winter, 1999) and
rats (Zarrabian et al., 1999). These factors may regulate
APN via mRNA abundance as well as protein expres-
sion. Indeed, in vivo experiments showed in indirect
measurements that mammary tissue has the ability to
overcome a limitation in availability of a single AA
(such as His or Lys) by increasing blood flow, uptake,
and extraction of AA from blood in both forms of FAA
and PBAA (Bequette et al., 2000; Mabjeesh et al., 2000).
Therefore, APN may play a role in milk protein synthe-
sis and secretion. This would be similar to the role of
γ-GT, which plays an important role in milk protein
production in the ovine lactating mammary tissue
(Johnston et al., 2004). Inhibition of γ-GT by incubating
the tissues with specific inhibitor (acivicin) decreased
milk protein secretion by 75%.
In conclusion, APN and other peptidases such as γ-
GT are candidates for active involvement in the mam-
mary gland to support protein synthesis and milk pro-
duction. Expression and activity of these enzymes
might be orchestrated in accordance to the nutritional
and physiological conditions. From the current study,
it appears that APN expression may be partly con-
trolled by the metabolic requirements of the gland and
postabsorptive forms of AA in the circulation.
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