Conference: Molecular and Cellular Studies of Rumen

Epithelial Metabolism

Use of Isolated Ruminal Epithelial Cells in the Study of Rumen Metabolism1

R. L. Baldwin VI
Nutrient Metabolism and Conservation Laboratory, USDA, ARS Livestock and Poultry Sciences Institute,
Beltsville Agricultural Research Center, Beltsville, MD 20705

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ABSTRACT A comprehensive understanding of ruminal development and metabolism has not yet been achieved.
The study of rumen epithelial metabolism during development can facilitate the development of feeding strategies
for developing pre-ruminant animals and mature animals. Understanding the effect of the physical form and
nutrient composition of the diet on the ruminal epithelium will lead to changes in dietary regimens that exploit
beneficial tissue responses. Characterization of the ontogenic shifts in ruminal metabolism, in association with
the description of physical changes, has established more discrete periods during the development of the ruminal
epithelium for future studies to be conducted. Isolated ruminal epithelial cells, specifically cells of the strata basale
and spinosum, have been used for metabolic studies of rumen epithelial energy metabolism. Because the ruminal
epithelium is a major producer of ketone bodies in the fed ruminant animal, it is integral to the energy metabolism
of the whole animal. Arguably, whole tissue slices may provide better estimations of actual tissue performance;
however, the benefits gained by maintaining tissue integrity are offset because of the high variability in tissue
composition due to dietary influences. Use of enriched cell populations is ideal for short-term incubations and
provides high cell yields with limited delay following removal of the tissue from the animal. Although the ruminal
cell isolation system is continuously undergoing refinement, enriched cell cultures have provided realistic results
with respect to known responses in vivo. J. Nutr. 128: 293S–296S, 1998.

KEY WORDS: • rumen • metabolism • epithelium • isolated cells

METABOLISM OF THE RUMINAL EPITHELIUM
The rumen and reticulum account for more than 70% of
the total digestive tract volume in ruminants (Stobo et al.
1966). Vast numbers of papillae protrude from the ruminal
surface into the lumen, greatly increasing the surface area
available for absorption of volatile fatty acids (VFA) (Harfoot
1978), and thus a large majority (ú75%; Church 1975) of the
VFA are absorbed through the epithelial lining of the rumen
and reticulum, with less than 10% of the VFA passing into
the small intestine (Harfoot 1978). In fact, most of the host
ruminant animal’s energy requirements are met by the VFA
absorbed through the epithelial lining of the rumen (Annison
and Armstrong 1969). Ruminal epithelium is not simply a
barrier to nutrient diffusion, but through the metabolism of
VFA, primarily butyrate, it acts to maintain metabolite con-
centration gradients and facilitate nutrient absorption.
The primary ruminal volatile fatty acids (acetate, propio-
nate and butyrate) are metabolized to different extents by the
ruminal epithelium. Experiments in vitro demonstrated that
1
Presented as part of the 38th Annual Ruminant Nutrition Conference ‘‘Molec-
ular and Cellular Studies of Rumen Epithelial Metabolism’’ given at the Experimen-
tal Biology 97 meeting, April 6, 1997, New Orleans, LA. This conference was
sponsored by the American Society for Nutritional Sciences and was supported
in part by educational grants from Cargill-Animal Nutrition Division, Eli Lilly and
Co., Monsanto, Moorman Manufacturing Co. and Purina Mills, Inc. Guest editor
for the conference publication was Barry W. Jesse, Cook College Rutgers Univer-
sity, New Brunswick, NJ.
although acetate is absorbed readily by the ruminal epithelium,
it fails to induce an increase in oxygen uptake by ruminal
epithelial tissue slices (Goosen 1976), thus indicating that the
tissue does not metabolize acetate to a great extent. Propionate
is metabolized to lactate and pyruvate by the ruminal epithe-
lium, with estimates ranging from 3% to 15% (Emmanuel
1981, Nocek et al. 1980, Weekes 1974a, Weigand et al. 1975),
whereas addition of propionate in vitro to ruminal slices re-
sulted in no change in oxygen uptake by the ruminal pieces
(Goosen 1976). Despite the apparently low use of propionate
by the ruminal epithelium, disappearance of propionate carbon
from the rumen cannot be completely accounted for by lactate,
pyruvate and propionate appearance in the portal blood.
In the fed ruminant animal, the rumen and the liver are
the primary producers of ketone bodies (Heitmann et al. 1987).
Ruminally derived acetoacetate (AcAc) is quantitatively re-
moved from portal blood by the liver and metabolized to b-
hydroxybutyrate (bHBA). Thus, in the fed state, the ruminal
epithelium is the primary source of circulating ketone bodies
in the ruminant animal (Heitmann et al. 1987). Most (85%
to 90%) of ruminally absorbed butyrate carbon appearing in
portal blood is in the form of bHBA and AcAc (Beck et al.
1984). The balance of the absorbed butyrate carbon can be
accounted for as CO2 produced as a result of butyrate oxidation
(Goosen 1976).
Ketogenesis in the ruminal epithelium is performed exclu-
sively in the mitochondria (Leighton et al. 1983) due to the

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294S SUPPLEMENT
compartmentalization of the ketogenic enzymes. Two path-
ways of ketogenesis are available to the ruminal epithelium.
Ketogenesis can progress through 3-hydroxy-3-methylglutaryl-
CoA (HMG-CoA) synthase and HMG-CoA lyase, as in the
liver, or via deacylation of AcAc-CoA catalyzed by succinyl-
CoA transferase. Both of these pathways result in the produc-
tion of AcAc. The final step in ruminal ketogenesis is the
production of bHBA catalyzed by bHBA dehydrogenase. In
the fed ruminant, this pathway is favored because of the
NADH:NAD ratio within the mitochondria (Heitmann et al.
1987). There are sufficient activities of these ketogenic en-
zymes present within the ruminal epithelium to account for
the ketogenic capacity of the rumen (Baird et al. 1970, Bush
and Milligan 1971, Leighton et al. 1983).
STRUCTURE OF THE RUMINAL EPITHELIUM
The rumen epithelium is responsible for physiologically im-
portant functions, such as absorption, transport, VFA metabo-
lism and protection (Gálfi et al. 1991, Stevens 1969). The
ruminal epithelium is a stratified squamous epithelium con-
sisting of four strata: stratum basale, stratum spinosum, stratum
granulosum, and stratum corneum (Steven and Marshall
1969). The stratum basale is the layer of cells immediately
adjacent to the basal lamina, and these cells contain fully
functional mitochondria and other organelles. The intermedi-
ate cell layers are the stratum spinosum and stratum granulo-
sum, which are not separated by a distinct division (Stevens
1969). As cells migrate through these intermediate layers, they
contain progressively fewer numbers of mitochondria and take
on a less uniform appearance. Thus, the cells of the strata
basale and spinosum, which have significant numbers of mito-
chondria, contribute most to the metabolic properties of the
tissue, i.e., ketogenesis. Consequently, these cells are the most
important cells of the rumen with regard to whole-animal
energy metabolism. In the stratum granulosum, the cells have
established tight gap junctions (desmosomes) that act as a
barrier to diffusion across the rumen wall (Steven and Marshall
1969). Cells in the stratum corneum, the most external cell
strata, are highly cornified, and presumably function as a defen-
sive barrier against the physical environment of the rumen.
The desmosomal integrity of the stratum corneum has degener-
ated, and large gaps exist between individual cells (Steven and
Marshall 1969). The number of cell layers present in the stra-
tum corneum is highly related to the dietary composition
(Gaebel et al. 1987). High concentrate diets, decreased rumi-
nal pH, increased propionate:acetate ratios, and increased mo-
lar proportions of butyrate all result in a stratum corneum of
up to 15 cell layers in thickness (Gaebel et al. 1987). Con-
versely, in an animal maintained on a high roughage diet the
stratum corneum may consist of as few as four cell layers
(Gaebel et al. 1987). Feeding regimen (i.e., how and when
the feed is presented) has also been implicated as affecting the
stratum corneum (Sakata and Tamate 1974, Tamate et al.
1974), which is most likely attributable to the subsequent
alterations in the ruminal environment (Gálfi et al. 1991).
Because of the unique and variable physical composition of
the ruminal epithelium, it has been difficult to investigate
the effects of differing ruminal environments on all aspects of
ruminal metabolism. Use of isolated ruminal epithelial cells
affords several advantages in the study of ruminal metabolism
and development. Isolation of the metabolically active cells
of the strata basale and spinosum minimizes the effects of
increased strata corneum and granulosum components when
evaluating metabolism of ruminal epithelium isolated from
animals fed diets differing in energy density (i.e., forages vs.
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concentrates) and ruminal characteristics. Furthermore, use of
isolated cells is necessary in order to compare data from the
undeveloped ruminal epithelium, consisting primarily of strata
basale and spinosum cell types, and fully differentiated ruminal
epithelium, consisting of four strata with highly variable cell
content. Thus, comparisons between mature and neonatal ru-
minal epithelial metabolic activities are difficult to interpret
when results can be expressed only upon a total tissue dry
weight basis or tissue protein basis. Only through investigation
of cells actively metabolizing VFA, i.e., cells of the strata basale
and strata spinosa, from the neonatal and mature rumens can
valid comparisons of cellular capabilities be made. Significant
limitations exist with cell isolation techniques, such as the
disruption of cell to cell interactions, and need to be addressed
when results of isolated cell preparations are to be extended
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to the whole tissue or whole-animal level.
DEVELOPMENT OF ISOLATED CELL SYSTEMS
Ruminal epithelial cell isolation systems have been devel-
oped by a number of groups for various purposes. Weekes
(1974b) reported that epithelial propionate metabolism to lac-
tate and pyruvate was severely compromised when bovine ru-
minal papillae were pre-treated and incubated with papain to
disrupt the tissue. Despite the isolation of apparently healthy
cells, Weekes (1974b) demonstrated that the activities of lac-
tate dehydrogenase, glutamate dehydrogenase, bHBA dehy-
drogenase, and NADP-malate dehydrogenase were reduced as
a result of papain treatment. Gálfi et al. (1980) demonstrated
through the use of a serial trypsinization procedure that bovine
ruminal cells, primarily from the strata basale and spinosum,
could be isolated and maintained in primary culture. Using
this isolation and primary culture system, these researchers
were able to demonstrate keratinization of ruminal cells in
culture (Gálfi and Néogrady 1989), an inhibitory role of buty-
rate (10 mmol/L) on DNA synthesis (Gálfi et al. 1981, Néo-
grady et al. 1989) and stimulatory effects of insulin on ruminal
cell proliferation (Néogrady et al. 1989). However, this proce-
dure was severely limited for use in metabolic evaluations in
that it required approximately 8 h to isolate the cells from the
strata basale and spinosum. Inooka et al. (1984) were able to
successfully isolate and culture ruminal cells using a pretrypsin-
isation procedure that facilitated isolation of both epithelial
and fibroblastic cell types by subsequent trypsinization in ap-
proximately 2 h. Inooka et al. (1986) were further able to
maintain and subculture isolated ruminal cells that had fibro-
blast characteristics. To facilitate the study of ruminal epithe-
lial metabolism during development and under conditions
where ruminal morphology was affected, Baldwin and Jesse
(1991) developed an isolation procedure that also used serial
trypsinization and resulted in a yield of viable cells sufficient
to conduct short-term (2-h) incubations with multiple treat-
ments within 2 h following removal from the animal. Baldwin
and Jesse (1991) reported linear rates of bHBA production
through 2-h incubations, demonstrated that chilling the rumi-
nal epithelium before isolating cells depressed subsequent keto-
genic capacity, and established that cells from the initial incu-
bation fractions were primarily of stratum corneum origin
through histological analysis of the digested tissue.
METABOLISM STUDIES USING ISOLATED
RUMINAL EPITHELIAL CELLS
The rumen is incompletely developed both physically and
metabolically at birth, representing only 30% of the total gas-
trointestinal capacity (Warner et al. 1956). Rumens of neo-
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 ISOLATED RUMINAL EPITHELIAL CELLS
nates do not exhibit the high degree of keratinization charac-
teristic of the mature organ. Metabolically, the rumen of neo-
nates is essentially nonfunctional with respect to ketogenic
capacity (Warner et al. 1956). It has been assumed that glucose
is the primary energy substrate of the immature tissue, as is
the case with other neonatal tissues (White and Leng 1980).
However, Baldwin and Jesse (1992) demonstrated, using iso-
lated ruminal epithelial cells from normally reared lambs rang-
ing from 0 to 56 d of age, that both butyrate and glucose were
used by the pre-ruminant preparations in vitro. The pattern
of glucose use by ruminal epithelial cells reported by Baldwin
and Jesse (1992) is consistent with previous experiments in
vitro using oxygen uptake by rumen slices from 14-d-old calf
ruminal epithelium (undeveloped) or mature ruminal papillae
(Giesecke et al. 1979). Oxygen uptake by the neonatal rumen
was greatest when glucose was present as the oxidizable sub-
strate. Although oxygen consumption by mature ruminal pa-
pillae increased above basal oxygen uptake when glucose was
added, the response was not as dramatic as observed with
neonatal ruminal slices (Giesecke et al. 1979). However, both
glucose and butyrate were oxidized to carbon dioxide by iso-
lated ruminal cells as early as 7 d of age (Baldwin and Jesse
1992). The rate of metabolism, on a cellular basis, declined
for butyrate as the animals matured, whereas glucose remained
elevated through 42 d and thereafter was markedly reduced
(Baldwin and Jesse 1992). The presence of butyrate in the
medium decreased the rate of glucose use by the isolated cells,
and butyrate oxidation was similarly affected by glucose (Bald-
win and Jesse 1992). Thus, the neonatal ruminal epithelium
can apparently use either butyrate or glucose, depending upon
supply of nutrients and cellular requirements.
A 10-fold increase in bHBA production by isolated cells
occurred between d 42 and 56, with no change in bHBA
dehydrogenase activity (Baldwin and Jesse 1992). Lane (1996)
conducted experiments that demonstrated that the pattern of
bHBA production observed in conventionally reared sheep
was similar to that observed in lambs maintained solely on
milk replacer through 84 d of age. Furthermore, Lane (1996)
demonstrated that ketogenic enzyme mRNA concentrations,
specifically 3-hydroxy-3-methylglutaryl-CoA synthase and
acetoacetyl-CoA thiolase, increased with increasing age in
both conventionally reared and milk replacer–fed lambs. Vol-
atile fatty acid infusions into the rumen of milk replacer–fed
lambs resulted in increased AcAc production and decreased
glucose oxidation by isolated ruminal epithelial cells (Lane and
Jesse 1997). Thus, metabolic adaptations in the developing
ruminal epithelium seem to be related to age and the presence
of luminal VFA.
Metabolite interactions have also been investigated using
isolated ruminal epithelial cells. Baldwin and Jesse (1991) re-
ported increased bHBA production from butyrate (10 mmol)
by isolated ruminal epithelial cells when physiological (25
mmol) concentrations of propionate were included in the in-
cubation medium, whereas acetate (50 mmol) inhibited
bHBA production. In further experiments, propionate stimu-
lation of butyrate oxidation to bHBA was found to be highly
correlated with lactate production (r2 Å 0.78; Baldwin and
Jesse 1996). Production of AcAc was decreased in these experi-
ments such that total ketone body production was unaffected
by propionate, whereas the ratio of bHBA to AcAc increased
from 1.2 to 6.22 with propionate concentrations from 0 to 50
mmols/L. Addition of succinate resulted in comparable lactate
production, yet inhibited bHBA production and none of the
additions (succinate or propionate) affected butyrate oxidation
to carbon dioxide. Thus, Baldwin and Jesse (1996) concluded
that the mitochondrial redox status of the ruminal epithelial
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295S
cell is affected by the presence of propionate such that com-
plete conversion of AcAc to bHBA is favored. This is consis-
tent with previous work with ruminal papillae in short-term
culture, which demonstrated that changes in redox state affect
the ratio of ketones produced (Emmanuel 1981, Goosen 1976).
Additional evidence that substrate level interactions influence
ruminal metabolism was reported by Jesse et al. (1992), who
showed that palmitate metabolism was more sensitive to rumi-
nally derived factors than to blood-borne factors. Jesse et al.
(1992) also demonstrated that rates of palmitate oxidation to
carbon dioxide by isolated ruminal cells were lower in neonatal
lambs (3–4 d) than in 24- to 30-wk-old lambs with mature
ruminal epithelium.
Very few of the studies conducted to evaluate changes in
ruminal epithelial metabolism in response to alterations in
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dietary regimen (i.e., forage vs. concentrate) have been able
to demonstrate metabolic changes (Harmon 1986). Harmon
(1986) reported that ruminal papillae from animals fed con-
centrate at 6.3 MJ ME/d oxidized both glucose (5 mmol/L)
and glutamine (1 mmols/L) to CO2 at higher rates than papil-
lae from steers fed forage at 3.4 MJ ME/d. Baldwin and McLeod
(1997) incubated ruminal epithelial cells isolated from lambs
fed either forage- or concentrate-based diets at two levels of
energy intake in the presence of multiple concentrations of
oxidizable substrates (acetate, propionate, butyrate, glucose,
glutamine and glutamate) in order to facilitate the develop-
ment of estimates of maximal oxidative capacity (Vmax) as
well as the dose at which half-maximal oxidative rates were
achieved (Kox). They found that oxidation rates of butyrate
and propionate were greater in epithelial cells isolated from
concentrate-fed lambs as compared with those isolated from
forage-fed lambs at some concentrations, whereas rates of ace-
tate oxidation were generally enhanced with increasing ME
intake. However, these changes in VFA oxidation rates did
not affect the overall predicted Michealis-Menton parameter
estimates, Kox and Vmax . The observed rates of oxidation re-
ported by Baldwin and McLeod (1997) were generally consis-
tent with the literature derived from research on ruminal tissue
pieces. Furthermore, the estimated Kox values reported are con-
sistent with the contention that VFA are the primary oxidiz-
able fuels used by ruminal epithelium (0.4, 0.9 and 9.9 mmol/
L for butyrate, propionate and acetate, respectively). In fact,
at ruminal concentrations, all of these metabolites would be
oxidized by the ruminal cells at maximal rates. This raises the
question of whether the concentration of the VFA surrounding
the strata basal and spinosum ever reaches such high concen-
trations in vivo. Possibly, through the protection of the outer
strata, rapid diffusion into the blood, and metabolism by the
ruminal epithelial cells themselves, VFA may be maintained
at lower concentrations. Clearly, more research needs to be
conducted to further refine these estimates and facilitate the
accurate prediction of the effect of dietary manipulation on
ruminal metabolism in vivo.
FUTURE RESEARCH
Isolation of cells from ruminal papillae biopsies has been
reported (Waldron et al. 1995) and represents a significant
improvement upon previously developed models. Harvesting
of tissue in this manner will facilitate research into the effects
of physiological status, as well as allow for the definition of
temporal responses of ruminal metabolism during transitions
between diets. The effect of alterations in ruminal pH and
VFA concentration also needs to be studied in more detail
using both in vivo and in vitro perturbations to refine our
understanding of the role of the ruminal epithelial metabolism
in the energy metabolism of the whole animal.
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