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THE EFFECTS OF DEXAMETHASONE AND SUNFLOWER
OIL ON ADIPOSE TISSUE DEVELOPMENT
IN BEEF STEERS

presented by

Matthew Pierce McCurdy

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6/01 cJClRCJDatoDuopes-pJS

THE EFFECTS OF DEXAMETHASONE AND SUNFLOWER
OIL ON ADIPOSE TISSUE DEVELOPMENT
IN BEEF STEERS
By

Matthew Pierce McCurdy

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

MASTER OF SCIENCE
Department of Animal Science

2003

ABSTRACT

THE EFFECTS OF DEXAMETHASONE AND SUNFLOWER OIL ON
ADIPOSE TISSUE DEVELOPMENT IN BEEF STEERS

By

Matthew P. McCurdy

Experiments were conducted investigating the development of
subcutaneous and intramuscular adipose tissue in beef steers in response
to glucocorticoid. In the first experiment, twenty-four early weaned steers
were randomly assigned to either a basal diet or a basal diet with 8%
sunflower oil and intramuscular injections every 28 d of either
dexamethasone or saline for a period of 112 d. There were no significant
differences in carcass characteristics due to treatment. In the second
experiment, four steers received either a single injection of saline or
dexamethasone 24 h before harvest. Total RNA was extracted from
subcutaneous and intramuscular adipose tissue. Analysis by cDNA
microarray revealed that DEX up-regulated (P < 0.01) expression of
urokinase receptor in both adipose depots, and that intramuscular adipose
exhibited significantly greater expression of genes involved in cellular
proliferation. Northern blot analysis revealed glucocorticoid receptor (GR)
expression was lower (P < 0.01) in intramuscular than in subcutaneous
adipose depots and administration of DEX lowered GR in both adipose
depots (P < 0.05). Differential gene expression between depots may

provide a way for manipulation of adipose tissue accumulation.

ACKNOWLEDGMENTS

I would first like to express my thanks to Drs. Burton, Doumit, and
Herdt for serving on my graduate committee. I also wish to express my
gratitude to Dr. Dan Buskirk for serving as my major professor. His
flexibility in allowing me to be involved in teaching, research, extension,
and extracurricular opportunities was greatly appreciated. Most of all I
would like to thank him for being a mentor and for always expecting the
best

I wish to thank Ken Metz and the crew at the Beef Cattle Teaching
and Research Center for all of their assistance as well as Joel Cowley for
his many hours of ultrasound work. I would also like to thank Dr. Paul
Coussens, Sue Sipkovsky, and the group in the Center for Animal
Functional Genomics for their training and assistance in the completion of
my project. Also I would like to extend a very special thanks to Dr. Patty
Weber and the crew in the lmmunogenetics Lab for training me on
countless procedures and for their ever-positive attitude.

While attending MSU my deepest appreciation has been for my
peers around me. I can truly say that a more talented group of graduate
students and staff has probably never been brought together from around
the country. To have been able to work with such a group has been a

humbling experience and an honor.

iii

I wish to thank my family, all of whom have supported me in my
every endeavor, as well as all of my friends, both here and other places
that have always been there to pull me through. Last, but far from least, I
owe a debt of gratitude to the people who instilled in me a love for the beef
business and a desire to set upon my chosen career path: the ranchers

and cattlemen of Dewitt County, Texas, and my father, one of them.

iv

TABLE OF CONTENTS

LIST OF TABLES ................................................................................ vi
LIST OF FIGURES ............................................................................. vii
INTRODUCTION ................................................................................. 1
CHAPTER |

Review of Literature

Beef quality, marbling, and palatability ................................................ 4
Implications of subcutaneous fat accumulation ................................... 8
Comparative biology of adipose tissue depots .................................. 1 0
Origin of adipose cells ....................................................................... 1 6
Adipocyte differentiation .................................................................... 1 7
Hormones and signaling in adipose tissue ........................................ 2 0
Glucocorticoids .................................................................................. 2 3
Fatty acids ......................................................................................... 2 6
cDNA microarray analysis ................................................................. 2 7
Literature cited ................................................................................... 3 0
CHAPTER II

Influence of supplemental sunflower oil and dexamethasone
therapy on performance and adipose tissue development in
early weaned beef steers

Abstract ............................................................................................. 3 9
Introduction ........................................................................................ 4 1
Materials and methods ...................................................................... 4 3
Results and discussion ...................................................................... 50
Implications ........................................................................................ 5 5
Literature cited ................................................................................... 6 6
CHAPTER III

Differential gene expression of subcutaneous and intramucular
adipose tissue from beef steers treated with dexamethasone

Abstract ............................................................................................. 6 8
Introduction ........................................................................................ 7 0
Materials and methods ...................................................................... 7 2
Results and discussion ...................................................................... 7 8
Implications ........................................................................................ 8 3
Literature cited ................................................................................... 9 6
CHAPTER IV

Interpretive Summary ...................................................................... 9 9

Table 2-1.

Table 2-2.

Table 2-3.

Table 2-4.

Table 2-5.

Table 2-6.

Table 2-7.

Table 3-1.

LIST OF TABLES

Composition of finishing diets .............................................. 56

Fatty acid profile of sunflower oil provided to steers
during the112 d treatment period ........................................ 57

Finishing performance of steers that received saline

(SAL) or dexamethasone (DEX) treatments and

received diets containing no supplemental oil (CON) or
sunflower oil (OlL) for a 112-d treatment period ................. 58

Ultrasound measures of steers that received saline

(SAL) or dexamethasone (DEX) treatments and

received diets containing no supplemental oil (CON) or
sunflower oil (OIL) for a 112 d treatment period .................. 59

Carcass data for steers that received saline (SAL) or
dexamethasone (DEX) treatments and received diets
containing no supplemental oil (CON) or sunflower oil

(OIL) for a 112 d treatment period ...................................... 60

Proximate analysis of longissimus dorsi muscle from rib
steaks of steers that received saline (SAL) or
dexamethasone (DEX) treatments and received diets
containing no supplemental oil (CON) or sunflower oil

(OIL) for a 112 d treatment period ...................................... 61

Fatty acids as a percentage of total fatty acids for steers

that received saline (SAL) or dexamethasone (DEX)
treatments and received diets containing no

supplemental oil (CON) or sunflower oil (OIL) for a 112

d treatment period ............................................................... 62

Densitometer quantitations of northern blots ....................... 84

vi

Figure 2-1

Figure 2-2.

Figure 2—3.

Figure 3-1.

Figure 3-2.

Figure 3-3.

Figure 3-4.

Figure 3-5.

LIST OF FIGURES

Ultrasound readings of intramuscular fat taken at 28 d
increments for steers that were control, received
supplemental sunflower oil in the diet (OIL), received
treatments of dexamethasone (DEX), or received
supplemental sunflower oil in the diet and received
treatments of dexamethasone (OIL+DEX) .......................... 63

Ultrasound readings of subcutaneous 12th rib fat taken

at 28 d increments for steers that were control, received
supplemental sunflower oil in the diet (OIL), received
treatments of dexamethasone (DEX), or received
supplemental sunflower oil in the diet and received
treatments of dexamethasone (OIL+DEX) .......................... 64

Ultrasound readings of subcutaneous rump fat taken at

28 d increments for steers that were control, received
supplemental sunflower oil in the diet (OIL), received
treatments of dexamethasone (DEX), or received
supplemental sunflower oil in the diet and received
treatments of dexamethasone (OIL+DEX) .......................... 65

Northern blots: Glucocorticoid receptor 9-alpha subunit
mRNA (top); beta-actin (bottom) ......................................... 85

Densitomer quantifications of northern blots across
treatment group averages ................................................... 86

Expression of mRNA for urokinase receptor in

intramuscular (IM) and subcutaneous (SQ) adipose

tissue from steers treated with saline (CON) or
dexamethasone (DEX) ....................................................... 87

Expression of mRNA for hypoxanthine
phosphoribosyltransferase in intramuscular (IM) and
subcutaneous (SQ) adipose tissue from steers treated

with saline (CON) or dexamethasone (DEX) ...................... 88

Expression of mRNA for alpha tubulin in intramuscular

(IM) and subcutaneous (SQ) adipose tissue from steers
treated with saline (CON) or dexamethasone (DEX) .......... 89

vii

Figure 36 Expression of mRNA for MDM2 in intramuscular (IM)
and subcutaneous (SQ) adipose tissue from steers
treated with saline (CON) or dexamethasone (DEX) .......... 90

Figure 3-7. Expression of mRNA for apoptosis regulator bax-alpha
in intramuscular (IM) and subcutaneous (SQ) adipose
tissue from steers treated with saline (CON) or
dexamethasone (DEX) ....................................................... 91

Figure 3-8. Expression of mRNA for TRAF5 in intramuscular (IM)
and subcutaneous (SQ) adipose tissue from steers
treated with saline (CON) or dexamethasone (DEX) .......... 92

Figure 39 Expression of mRNA for serine protease inhibitor p19 in
intramuscular (IM) and subcutaneous (SQ) adipose
tissue from steers treated with saline (CON) or
dexamethasone (DEX) ....................................................... 93

Figure 3-10. Expression of mRNA for caspase-4 in intramuscular
(IM) and subcutaneous (SQ) adipose tissue from steers
treated with saline (CON) or dexamethasone (DEX) .......... 94

Figure 3-11. M-A scatterplot of data from array comparing
intramuscular, control tissue vs. intramuscular,
dexamethasone-treated tissue before and after
normalization ...................................................................... 95

viii

INTRODUCTION

Adipose tissue development in beef cattle production is an area
that poses a challenge to the beef industry. A way must be found to
reduce the fat content of meat carcasses to provide a nutritious product
with minimum waste fat, while not affecting meat palatability (Smith and
Crouse, 1984). Beef cuts with greater amounts of intramuscular fat or
marbling have a higher probability of being juicy and flavorful, and of
remaining tender when cooked to a greater degree of doneness. The
National Beef Quality Audit (Smith et al., 2000) revealed that only 19.3%
of the nation’s beef supply contained at least a “Modest" degree of
marbling as compared to a projected future consumer demand of 28%.

Within a carcass maturity classification, marbling is the major
determinant of USDA Quality Grade. Because of this, subtle differences
in the amount of visible intramuscular fat can dramatically change carcass
value. Subcutaneous fat, however, has been shown to be less beneficial
to the quality and palatability of beef and greater amounts of
subcutaneous fat result in greater trim waste from beef carcasses. Cattle
are often fed for extended periods of time to maximize marbling, because,
it is generally accepted that the sequence of adipose accumulation from
early to late maturity is: perirenal, subcutaneous, interrnuscular and
intramuscular. Cattle therefore often accumulate large amounts of

external fat before marbling can develop. In addition, extended feeding

periods come at the expense of declining efficiency of converting feed to
muscle protein.

Due to differences in the rate of accretion between subcutaneous
and intramuscular adipose depots, research has focused on examining
the two adipose depots on a cellular level. Evidence has accumulated to
indicate that intramuscular adipocytes exhibit different cellular properties
as opposed to more extensively investigated subcutaneous adipocytes.
Adipocytes from subcutaneous depots are larger and more spherical in
shape and contain larger quantities of triacylglycerides suggesting that
adipocytes in subcutaneous adipose mature at a faster rate and reach
terminal differentiation earlier than adipocytes in intramuscular depots.
Additionally, intramuscular and subcutaneous adipocytes exhibit metabolic
and physiological differences.

Glucocorticoids are routinely used to induce differentiation in
adipocytes in vitro. In live animals, glucocorticoids have been shown to
have a lipolytic effect in subcutaneous adipose stores. However,
glucocortcoids have enhanced or not affected intramuscular adipose
accumulation.

Due to the needs of the beef industry being in direct opposition of
normal adipose tissue depot development, it is imperative that the
molecular pathways that lead to development and terminal cellular
differentiation of adipocytes be explored and that biological differences

between adipose tissue depots be further characterized. Therefore, the

goal of the present study was to identify differences between
subcutaneous and intramuscular adipose tissue and to characterize the

differential response of the two depots to stimulation by a glucocorticoid.

CHAPTER I

REVIEW OF LITERATURE

Beef quality, marbling, and palatability

Importance of marbling. One of the greatest challenges facing the
beef industry is the lack of sufficient marbling in carcasses of beef cattle to
meet the demands of the consumer. Marbling is the amount and
distribution of visible intramuscular fat in the muscle. According to the
2000 National Beef Quality Audit (NBQA), insufficient marbling/ low USDA
Quality Grade ranked in the top five “Greatest Quality Challenges”
identified by producers, packers, and wholesalers/retailers (3, 5, and 1
respectively) (Smith et al., 2000). The 2000 NBQA identified that the ideal
Quality Grade consist for the US. beef industry in 2000 was 6% Prime,
27% Upper 2/3 Choice, 32% Low Choice, 35% Select, and 0% Standard.
For the 2000 NBQA, researchers collected data from 9,396 carcasses in
thirty packing plants from across the US. Quality Grade distribution from
the collected data was shown to be 2.0% Prime, 17.3% Upper 2/3 Choice,
31.8% Low Choice, 42.3% Select, and 6.5% Standard or hardboned. Due
to the shortcomings of the actual consist when compared to the ideal
consist, the estimated economic losses to the beef industry caused by
insufficient marbling were $20.96 for each fed steer and heifer harvested

in 2000.

The standards for USDA Quality Grades set forth in 1975 (USDA,
1975) state that the quality grade of a steer, heifer, cow, or bullock is
based on the evaluation of palatability-indicating characteristics of the
lean. Marbling continues to receive primary consideration in the
assessment of quality in the US. beef grading system (USDA, 1975;
Tatum et al., 1982), and thus within a carcass maturity classification,
marbling is the major determinant of USDA Quality Grade. In the current
US. grading system, this makes marbling the dominant indicator of beef
palatability.

Marbling and tendemess. Numerous studies have sought to link
marbling with tenderness and overall palatability. McBee and Wiles
(1967) reported that overall tenderness (determined by palatability panel
and shear force), juiciness, and flavor increased with additional degrees of
marbling in a linear fashion. Tatum et al. (1980) also reported an increase
in palatability of steaks as marbling score increased. Additionally, Dolezal
et al. (1982) found that marbling score was positively correlated with
increased sensory panel ratings and decreased shear force values. Berry
(1993) found that decreased shear force values existed for loin steaks
from carcasses with a higher marbling classification.

Smith and Carpenter (1974) reviewed the literature concerning the
link between fat content of meat and palatability. Additionally, they
attempted to elucidate the theories behind the effects of marbling on beef

palatability. Of the four theories presented in the paper, the first was the

bite theory. This theory suggests that with an increase in marbling, protein
is replaced by lipid in each bite size piece of meat thus lowering the bulk
density of each piece of meat. Fat has much less shear force resistance
than protein and a consequent increase in tenderness occurs. Another
theory presented was the lubrication theory. This idea states that
intramuscular fat provides lubrication for the muscle fibers that it
surrounds thus providing a juicier product. A third idea suggested is the
strain theory. The strain theory suggests that marbling physically aids the
tenderness of meat by disrupting the connective tissue structure of the
muscle fiber walls. This would provide a decrease in the strength and
thus the shear force resistance of the connective tissue associated with
the muscle fibers and bundles. This theory is given relevance by a much
more recent work by Nishimura et al. (1999) that evaluated changes in the
connective tissue of intramuscular fat in Japanese Black steers. When
examined by electron microscopy the longissimus muscle showed a
partially broken connective tissue structure where adipose tissue had
formed between muscle fiber bundles. Additionally, in the semitendinosus
muscle, where fat content in the muscle was significantly lower,
connective tissue structure remained rigid. It was concluded that the
intramuscular adipose tissue development aids beef tenderization by
disorganizing connective tissue structure (Nishimura et al., 1999). The
last of the theories presented by Smith and Carpenter (1974) is the

insurance theory. This theory concerns the tenderness of meat as it

relates to cooking method. The idea behind this theory is that increased
marbling in the meat allows for cooking at higher temperatures and dry-
heat methods of cooking. The fat tissue content allows the meat to
endure higher cooking temperatures, thus providing that meat that is
cooked by the wrong method or cooked too fast will still be palatable. The
work of Wheeler et al. (1999) supports this theory. Their study found that
steaks from carcasses that graded Top Choice (Upper 2/3 of the USDA
Choice grade) were less affected by elevated degree of doneness than
steaks from carcasses that graded Low Select. This suggests that beef
cuts with greater amounts of marbling have a higher probability of being
juicy and flavorful, especially when cooked well done by the consumer.
Smith et al. (1987) also reported that higher USDA Quality Grades were
associated with lessened variability in the palatability of cooked beef.
Relation to beef flavor. Other studies have reported that an
increase in marbling score and consequent increase in USDA Quality
Grade is associated less with tenderness and more with overall palatability
including flavor and juiciness. Francis et al. (1977) reported that between
rib-eye steaks that possessed either Slight or Modest levels of marbling,
flavor and juiciness of Modest marbled beef was significantly preferred by
a group of over eight-hundred consumers. Neely et al. (1998) conducted
a study on consumer satisfaction of beef. One component of the study
examined differences in consumer preference for USDA Quality Grades of

Top Choice, Low Choice, High Select, or Low Select. For overall like and

palatability consumers significantly preferred Top Choice to the other
USDA grades.

Again referring to the 2000 NBQA (Smith et al., 2000), it was
estimated that $38.30 per carcass was lost due to inadequate taste of
beef. It was further estimated that approximately 74% of the losses
associated with taste were caused by insufficient marbling.

Due to the reasons discussed above, marbling is not only the
primary determinant of USDA Quality Grade, but also of beef carcass
value. Because of this, subtle increases in the amount of visible
intramuscular fat can change carcass value by 10% or more, resulting in
greater revenues for all beef industry segments.

Implications of subcutaneous fat accumulation

Time-on-feed of beef cattle and subsequent accumulation of
subcutaneous fat has been evaluated as an indicator of palatability
attributes of beef by a number of studies. Dolezal et al. (1982) pointed out
that while the time-on-feed concept appears to be valid, the usefulness of
such a system would be limited by the complications of monitoring time-
on-feed of all cattle entering the supply chain. An adjunct to the time-on-
feed concept is a grading system based on the level of subcutaneous fat.

Subcutaneous fat and palatability. The fattening of animals has
historically been accepted as a means of increasing beef quality (Dolezal
et al., 1982). Smith et al. (1976) determined that subcutaneous fat aided

tenderness by slowing the process of carcass chilling in lamb and

protecting the muscle tissue from the effects of sarcomere shortening.
Tatum et al. (1982) investigated this effect on tenderness in beef and
determined that steaks from carcasses having a subcutaneous fat
thickness of less than 5.08 mm were lowest in palatability. However, there
seemed to be little improvement in palatability beyond 7.62 mm of
subcutaneous fat. It was concluded that fat thickness, as compared to
marbling, was ineffective as a predictor of cooked beef palatability.
Dolezal et al. (1982) compared subcutaneous fat thickness with marbling
as a predictor of beef palatability. Results from this study found that
steaks from carcasses with fat thickness of at least 5.08 mm were superior
in palatability to steaks with 5.07 mm or less. Similarly, Shackelford et al.
(1994) looked at the effect of a minimum fat thickness requirement of 5
mm on palatability within the USDA Select Quality Grade. It was found
that shear force values were lower and overall tenderness was higher for
carcasses that had fat thickness greater than 5 mm. The magnitude of the
differences in this study was small, however, and it was concluded that a
minimum fat thickness requirement for the Select grade would not greatly
improve tenderness.

Even if significant differences in palatability exist for beef carcasses
above or below the 5.08 mm level as indicated by Dolezal et al. (1982),
the usefulness of such a predictor may be very limited. The 2000 National
Beef Quality Audit showed that only 4.9% of the 9,396 carcasses

evaluated had less than 5.08 mm (0.2 inches) of subcutaneous fat

thickness (Smith et al., 2000). With over 95% of the nation’s beef supply
exceeding this level, the contribution of subcutaneous fat thickness to beef
palatability may already be close to maximization. Thus, the issues and
concerns related to subcutaneous adipose development in beef become
that of excess.

Economic losses. As stated previously, the insufficiency of
marbling and tenderness ranked in the top five concerns of producers,
packers and wholesalers/retailers as determined by the 2000 NBQA. In
the same listing of concerns was the problem of excess external fat
accumulation. It was estimated that $50.96 for each steer and heifer
harvested in 2000 was lost due to excess fat. Considering that
approximately 36.4 million cattle were harvested in the US. in 2000
(USDA, 2000), this translates to over 1.8 billion dollars in lost revenue
annually. Therefore, in contrast to marbling, it is the reduction of
subcutaneous fat that holds the key to increased profitability for the beef
industry.

Comparative biology of adipose tissue depots

The current beef industry faces a difficult challenge in altering the
fat content of its product. Because the desired future product requires a
reduction in subcutaneous adipose tissue and an increase in the amount
of intramuscular adipose tissue, the needs of the industry run in opposing

directions. Thus, the goals of altering beef to achieve the desired fat

10

content can only be met if differences exist in adipose tissue due to
location.

Growth and Development. As previously stated, fattening of cattle
has historically been accepted as a way to improve the palatability of beef
(Dolezal et al., 1982). It is also generally accepted that increasing
subcutaneous fat in cattle to certain end points will result in adequate
intramuscular fat levels. According to Hood (1983) fat is preferentially
deposited in the following tissue sites: perirenal and omental >
subcutaneous > interrnuscular > intramuscular. However, the relationship
between accumulation of subcutaneous and intramuscular adipose has
been shown to be inconsistent (Zinn et al., 1970). Dubeski et al. (1997)
conducted a study where cattle were fed to extreme slaughter weights. It
was observed that while the heavy slaughter weights (680 kg) produced
carcasses with extremely high amounts of external fat, they did not result
in highly marbled beef. Cunha (1974) observed that many cattle reach the
USDA Choice Quality Grade with only 0.2 to 0.3 inches of backfat and the
2000 NBQA determined that cattle grading USDA Choice spanned USDA
Yield Grades 1 to 5.

In 1984, Smith and Crouse observed that previous studies
indicated that marbling scores were less subject to manipulation through
nutrition as compared to backfat thickness or total carcass fat. In this
study, cattle fed a higher energy corn diet did not display significantly

greater marbling scores than cattle fed a lower energy corn silage diet.

11

Cattle on the higher energy diet did, however, have greater backfat
thickness and a greater percentage of kidney, pelvic, and heart fat. It was
thus concluded that lipogenesis in the two depots was controlled by
different means. Because adipose tissue metabolism appears to be
regulated differentially due to location, it is necessary to examine
differences between the two depots on a cellular and molecular level.
Cellulan'ty. It has been observed that intramuscular adipocytes
represent a unique cell population that exhibits different cellular and
metabolic activity as compared to adipocytes from subcutaneous tissue
(Lee and Kauffman, 1974; Lin et al., 1992). A number of studies have
pointed to the fact that intramuscular adipocytes are smaller cells on
average, with less cell volume and correspondingly less lipid content than
subcutaneous adipocytes (Hood and Allen, 1973; Smith and Crouse,
1984; Miller et al., 1991; May et al., 1994). Hood and Allen (1973)
observed that adipose tissue development in beef cattle was achieved by
both hyperplasia and hypertrophy of adipocytes. After 14 months of age,
cells in the subcutaneous adipose tissue had ceased to proliferate and
developed solely by hypertrophy. However, intramuscular adipose tissue
contained smaller cells (< 70 mm in diameter) that continued to actively
engage in hyperplasia throughout the study. Similarly, Lee and Kauffman
(1974) saw that hyperplasia in porcine intramuscular tissue never
plateaued throughout the 24 wk duration of their trial. In cattle, as in other

ruminant species, the majority of de novo fatty acid synthesis takes place

12

in adipose tissue. The smaller size and volume of adipocytes in the
intramuscular adipose is associated with decreased lipogenic activity.
Hood and Allen (1978) concluded that lipid was synthesized at a slower
rate in intramuscular adipose than in subcutaneous adipose and that this
was directly related to a smaller adipocyte volume.

Substrate utilization. In addition to differences in total lipogenic
activity, differences exist between subcutaneous and intramuscular
adipose tissue depots in their preferential uptake of substrates for fatty
acid synthesis. Smith and Crouse (1984) demonstrated that in explants of
bovine adipose tissue, subcutaneous adipocytes primarily used acetate-
derived substrates as precursors for fatty acid synthesis. However,
glucose-derived carbon units provided the main source of substrate for
fatty acid synthesis in intramuscular adipose tissue. It was observed that
acetate provided 70 to 80% of the acetyl units for lipogenesis in
subcutaneous adipose tissue but only 10 to 25% for intramuscular tissue.
Glucose provided only 1 to 10% of the acetyl units for fatty acid synthesis
in subcutaneous adipose but provided 50 to 75% of the units for
intramuscular fatty acid synthesis. Differences in esterification of
exogenous fatty acids have also been demonstrated between depots. Lin
et al. (1992) observed that subcutaneous adipose tissue more actively
esterified fatty aids than intramuscular adipose. However, intramuscular
adipose esterified a larger amount of palmitate as a percentage of total

fatty acids.

13

Brown adipose tissue. In many species (notably excluding swine)
there exists at birth a population of adipocytes known as brown adipocytes
(Smith, 1995). Most of the adipose tissue in newborn ruminant species is
brown adipose tissue, although small amounts of white adipose tissue are
present (Alexander et al., 1975; Alexander, 1978; Martin et al., 1997).
Brown adipocytes are characterized by a profound abundance of
mitochondria and the presence of specific gene products, such as the
mitochondrial uncoupling protein (Casteilla et al., 1987; Klaus et al., 1991;
Cousin et al., 1992; Brandler et al., 1993; Smith, 1995). Brown adipose
tissue is present at birth and functions for thennogenesis in order to
provide warmth for newborns. Shortly after birth however, it disappears or
interconverts to white adipose tissue (Smith, 1995). Martin et al. (1999)
observed that white subcutaneous adipose tissue from Angus and
Brahman steers contained adipocytes with distinct brown adipocyte
morphology, suggesting an involution of brown adipose tissue to white
adipose tissue in utero. To our knowledge, data has not been reported to
demonstrate whether or not intramuscular adipocytes originate from brown
adipose tissue. Intramuscular adipocytes may have a unique origin as
compared to adipocytes contained in subcutaneous adipose tissue.

Differential gene expression. Considering the observed differences
between adipose tissue depots, it is reasonable to think that subcutaneous
and intramuscular adipose may exhibit unique aspects of gene

expression. It has been demonstrated in a number of studies by Northern

14

and Western blot analyses that differences do exist between the two
depots at a molecular level.

It is well established that glucocorticoid induced differentiation takes
place in the presence of insulin in experiments using in vitro cell culture
models (Martin et al., 1998). This requires insulin-responsive glucose
transporter (GLUT-4), which is considered to be a major glucose
transporter in muscles and adipose tissue (Aso et al., 1995). However, in
experiments using a clonal bovine intramuscular preadipocyte line, GLUT-
4 protein was not detectable (Aso et al., 1995). This suggests that the
differentiation of adipocytes in intramuscular tissue may take place
through different means as compared to other fat tissue depots.

Kim et al. (2000) conducted a study to evaluate the expression of
the obese gene (ob), which encodes for leptin, in fed and fasted states
among adipose tissue depots in the bovine. They observed that obese
mRNA was moderately expressed in subcutaneous and interrnuscular
adipose, and that fasting effectively decreased levels of transcript
expression in these depots. In contrast, the obese gene was expressed
only at very low levels in the intramuscular adipose and fasting did not
elicit an expression change in this depot.

With variable behavior of cells in adipose tissue reflecting different
locations and conditions, a thorough understanding of the growth and
metabolism of adipose cells is critical for differential manipulation of

subcutaneous and intramuscular adipose tissue accumulation.

15

Origin of adipose cells

In most species the development of white adipose tissue (WAT)
begins prior to birth, although the developmental origin of adipocytes from
embryonic cells is not well characterized (Gregoire, 1998). However, a
number of studies using cloned cell lines suggest that the adipocyte
lineage is derived from an embryonic stem cell precursor with the capacity
to differentiate into the mesoderrnal cell types of adipocytes,
chondrocytes, osteoblasts, and myocytes (Konieczny and Emerson,
1984). Ailhaud et al. (1992) states that the process of determination from
the multipotential stem cells leads to the formation of unipotential
adipoblasts. The adipoblasts then undergo commitment to form
preadipocytes. These are cells that express early markers of
differentiation but not late markers and have yet to begin lipid
accumulation. It has not been clarified whether adipoblasts remain after
birth or if only preadipocytes are present (Ailhaud et al., 1992).
Postnatally, as preadipocytes undergo the process of terminal
differentiation they begin to express late markers of adipose cell
development as well as begin the accumulation of triacylglycerides. As
terminal differentiation continues, very late markers of adipose
development are expressed and the accumulation of lipid becomes more
pronounced. Committed preadipocytes retain the ability to proliferate,
whereas fully differentiated cells lose the capacity for mitosis (Ailhaud et

al., 1992). The growth of WAT increases rapidly after birth as result of

16

both adipose cell size and number. Gregoire et al. (1998) states that the
potential to acquire new fat cells from fat cell precursors throughout the life
span is undisputed, suggesting the continued presence of preadipocytes.
However, as previously cited, in cattle (Hood and Allen, 1973), adipose
development appears to take place exclusively by an increase in cell size
after approximately 14 months of age in subcutaneous tissue.
Adipocyte differentiation

During the growth phase, preadipocytes from cloned cell lines or
from primary culture are morphologically similar to fibroblasts (Gregoire et
al., 1998). As stated above, the preadipose cell has the ability to
proliferate. However, for terminal differentiation to take place the cell must
withdraw from the proliferative cycle. Terminal adipocyte differentiation is
characterized by early, intermediate, and late markers of gene expression,
as well as by the accumulation of lipid within the cell. Much of the
available information regarding adipocyte differentiation comes from
preadipose cell culture models. The process of differentiation appears to
begin with the growth arrest of preadipose cells. Two transcription factors,
CCAAT/enhancer binding protein a (CIEBP a) and peroxisome
proliferator-activated receptor-y (PPAR-ry), shown to activate adipocyte
specific genes, also appear to have a role in the growth arrest required for
adipocyte differentiation (Altiok et al., 1997).

Molecular events. Following the growth arrest of preadipocytes in

cloned cell lines, committed cells undergo at least one round of mitosis

17

providing clonal expansion of committed cells. This has not, however,
been shown to be the case with primary cell cultures and may indicate that
the necessary clonal amplification for differentiation may have already
taken place in vivo (Entenmann and Hauner, 1996). Clonal expansion
appears to be a unique process as compared to preconfluent cell growth
as indicated by the inactivation of growth promoting transcription factors
by retinoblastoma proteins (pRB, p107, and p130) (Richon et al., 1997).
The retinoblastoma family of proteins is known to inactivate the E2F family
of transcription factors associated with cell growth and proliferation
(Mulligan and Jacks, 1998).

During growth and differentiation, changes in cellular gene
expression begin to take place. Lipoprotein lipase plays a key role in the
accumulation of triacylglycerides and has been revealed to be an early
marker of adipocyte differentiation (Ailhaud, 1996). Two families of
transcription factors mentioned previously, C/EBPs (c, B, and 6 isoforms)
and PPARs (0t and y isoforms) are expressed during the early stages of
adipocyte differentiation. Increased expression of C/EBP-B and C/EBP-6
appears to be followed by an increase in C/EBP-a, as well as PPAR-y,
which is associated with the activation of adipocyte specific genes (Altiok
et al., 1997). The transcription factor PPAR-y has been determined to
have two isoforms (1 and 2). The expression of PPAR-yZ, which is
specific to adipose tissue, appears to be a key step in the induction of

adipocyte differentiation (Li and Lazar, 2002).

18

As changes in gene expression are induced, preadipose cells begin
to lose their fibroblastic morphology and adopt a more spherical shape.
Preadipocyte factor-1 (pref-1), a preadipose cell-specific protein, exhibits
a dramatic decrease in expression during adipocyte differentiation and is
not detectable in mature fat cells. Pref-1 may be responsible for
maintenance of preadipocyte morphology and changes in cell shape
during the differentiation process may be due to its down-regulation (Smas
etaL,1993)

Enzymatic changes. As differentiation progresses in 3T3 cell
cultures, many of the genes associated with lipid metabolism begin to be
expressed. Lipoprotein lipase (LPL) and hormone sensitive lipase (HSL)
expression becomes evident as well as that of GLUT-4 and fatty acid
binding protein (aP2). In addition, acetyl-CoA carboxylase and fatty acid
synthetase, two key enzyme markers for lipogenesis, begin to function
(Ailhaud et al., 1992). As the enzymes necessary for lipogenesis
including ATP citrate lyase, malic enzyme, stearoyl-CoA desaturase
(SCD1), glycerol-3-phosphate acyltransferase, glycerol-3-phosphate
dehydrogenase, fatty acid synthase, and glyceraldehydes—3-phosphate
dehydrogenase begin to be expressed, triacylglyceride accumulation is
initiated and lipid droplets form within the cell (Spiegelman et al., 1983;

Paulauskis and Sul, 1988; Weiner et al., 1991).

19

Hormones and signaling in adipose

Hormonal effects. Growth hormone (GH) has known lipolytic activity
and is inhibitory to differentiation. However, GH stimulates insulin-like
growth factor-1 (lGF-1) gene transcription and lGF-1 is necessary for
adipocyte differentiation. Growth hormone has been shown to be
necessary for differentiation in cloned murine cell lines, possibly to
sensitize the cells to mitogenic effects of lGF-1 (Corin et al., 1990).
However, this has not been shown to be necessary in primary cultures of
preadipocytes from rats (Wabitsch et al., 1995) and this may be attributed
to the prior in vivo exposure of primary culture cells to GH (Gregoire et al.,
1998)

lGF-1 is a requirement for adipocyte differentiation and has been
shown to stimulate adipogenesis in both cloned cell lines and primary
culture preadipocytes (Boney et al., 1994; Wabitsch et al., 1995). lGF-1
appears to be secreted from adipose cells and may act as an
autocrine/paracrine regulator of differentiation although the specific
mechanism of adipogenic stimulation remains unknown (Gregoire et al.,
1998)

Another important signaling hormone involved in adipose growth
and function is leptin. Leptin is secreted specifically by adipose tissue and
is thought to provide signals for regulation of body energy stores and
nutritional intake (Fried et al., 2000). Leptin is the protein product of the

ob gene, discovered by Zhang et al. (1994), and its expression is one of

20

the latest occurring markers of adipocyte differentiation (Gregoire et al.,
1998)

Other hormones that may influence differentiation include
triiodothyronine (T3) and catecholamines. Triiodothyronine appears to be
stimulatory to differentiation although the molecular mechanisms
underlying this effect are not known (Gaskins et al., 1989). Gharbi-Chihi
et al. (1981) observed, that T3 stimulation of differentiation is only evident
in certain cloned cell lines. Additionally, B-agonists or catecholamines are
known to affect adipocyte growth and metabolism. The B-agonists exert
their action through B-adrenergic receptors and are known to increase
lipolysis in adipocytes, both in vitro and in vivo (Mersmann, 1998). The [5-
agonists include the physiological compounds of norepinephrine and
epinephrine, as well as a large number of their synthetic analogs
(Mersmann, 1998). The B-adrenergic receptor has three subtypes (B1AR,
62AR, and B3AR) and differences in the distribution of the three subtypes
in adipose tissue vary according to stage of growth and development, as
well as between species (Mersmann, 1998). However, it has been
observed in in vitro cell cultures from 3T3 cloned cell lines that
preadipocytes have higher levels of the B1AR subtype. As the process of
adipocyte differentiation takes place, the expression of B1AR diminishes
and expression of [32AR and (33AR is up-regulated (Feve et al., 1990;

Feve et al., 1991).

21

Vitamins. Retinoic acid, the main active metabolite of Vitamin A
has been shown to play a key role in differentiation and is recognized as a
potent inhibitor of adipocyte differentiation (Villarroya et al., 1999).
Retinoic acid has been hypothesized to reduce the expression of early
gene markers in adipocyte differentiation including the C/EBP and PPAR
families of transcription factors (Villarroya, et al., 1999). Additionally,
vitamin C has been shown to enhance adipocyte differentiation in primary
cultures of bovine adipocytes (Yano et al., 2000). However, the mode of
action remains unknown.

Arachadonic acid metabolites. Various arachadonic acid
metabolites may also play a role in adipose tissue development and
significant amounts of prostaglandins (PGs) are excreted by mature
adipocytes from 3T3 cloned cell lines (Hyman et al., 1982). Prostacyclin
(PGlz) released from mature mouse adipocytes has been demonstrated to
have an adipogenic effect on neighboring preadipocytes by both the
elevation of levels of cAMP and intracellular free calcium (Ailhaud et al.,
1996) in the cell and as an activator of PPAR transcription factors (Brun et
al., 1996). While PGlz appears to act on 3T3 preadipocytes to induce
differentiation and has no effect on terminally differentiated adipose cells,
prostaglandin E2 (PGE2) appears to act the opposite way (Ailhaud et al.,
1996). Prostaglandin E2 shows no effect on preadipocytes but behaves
as an antilipolytic—hypertrophic effector of mature cells (Ailhaud et al.,

1996). Another PG, prostaglandin F20, (PGan) has been shown to inhibit

22

adipocyte differentiation in both cloned cell lines and primary culture cells
from multiple species and PGF20L is thought to act through a
calcium/calmodulin—dependent protein kinase, although the specific mode
of action is not well understood (Gregoire et al., 1998). Additionally,
prostaglandin J2, a derivative of the prostaglandin 02 has been shown to
be a PPAR-y agonist (Kliewer et al., 1995).

Additional signals. In addtion, two cytokines have been implicated
in adipose cell endocrinology. Tumor necrosis factor-a (TNF-a), a
mediator of apoptotis, and interleukin-6 (IL-6), a cytokine growth factor,
are secreted from mature adipocytes and appear to play a regulatory role
in adipose tissue growth and development by acting in a
paracrine/autocrine fashion (Morrison and Farmer, 2000).
Glucocorticoids

Effects on adipocytes. It has been well established that
glucocorticoids are a necessary part of the stimulation of differentiation in
adipocytes. In studies involving cloned cell lines, the glucocorticoid
analog dexamethasone has been observed to stimulate differentiation and
is routinely used as an inducer of differentiation on preadipocyte cell lines
in order to study mature adipocytes (Martin et al., 1998). Additionally,
dexamethasone has been shown to enhance differentiation in the
presence of insulin or serum in nearly every preadipocyte line studied
(Martin et al., 1998). Dexamethasone, has been shown to increase the

C/EBP-q isoform in embryonic mouse preadipocyes (Cao et al., 1991; Yeh

23

et al., 1995). This effect may be mediated through an interaction with the
retinoblastoma family of proteins (Mulligan and Jacks, 1998). Additionally,
it has been suggested by Wolf (2000) that the effects of the glucocorticoid
are mediated through a direct down-regulation of pref-1. In other studies
with mouse preadipocytes, glucocorticoid effects appear to be mediated
through increased metabolism of arachidonic acid leading to an increase
in production of prostacyclin, which in turn increases intracellular cAMP
(Ailhaud et al., 1992). Aubert et al. (2000) recently demonstrated that
activation of the prostacyclin receptor (IP receptor) via prostacyclin or
cyclic AMP-elevating agents was sufficient to rapidly up-regulate the earty
expression of both transcription factors C/EBP-a and C/EBP-S. These
observations are consistent with the idea that PGI2 is released from
preadipose cells and behaves as a paracrine/autocrine effector of adipose
cell differentiation.

Effects in live animals. Experiments involving glucocorticoids and
their effects in live animals are limited. There is however some evidence
to support the action of glucocorticoids on adipose tissue in vivo. Even
though adrenal glucocorticoids readily stimulate mobilization of lipid from
subcutaneous and other fatty depots, circulating levels of glucocorticoids
have been positively correlated with intramuscular fat deposition in cattle
(Cramer and Shahied, 1974).

Romans et al. (1974) observed that longissimus muscle lipid

increased as a result of induced exercise stress in 21-month-old steers. It

24

was observed that increases in exercise stress appeared to cause
lipolysis from adipose as evidenced by increases in circulating free fatty
acids, but did not have detrimental effects on intramuscular lipid. This
suggests that lipids may have been mobilized from specific adipose
tissues and redeposited in muscle.

Brethour (1972) showed that acute injections of dexamethasone
were effective at increasing intramuscular lipid when given from 30 to 90 d
before harvest. In steers that received 10 to 20 mg of dexamethasone at
90, 76, or 33 d preslaughter, significant increases were observed in
carcass marbling scores. Additionally, it was observed that marbling
scores were not increased in steers that received injections of
dexamethasone more than 90 d before harvest. Because steers were not
adapted to full feed until less than 90 d from harvest, it was concluded that
glucocorticoid effects may exhibit an interaction with body energy
reserves. Nevertheless, adipose from unique depots appeared to display
differential endocrine control in relation to glucocorticoids. A more recent
attempt to increase marbling using low dosage dexamethasone implants
was unsuccessful in cloned yearling Brangus steers (Corah et al., 1995).
Due to the implants slowed release over time (implants contained 100 mg
of dexmethasone), this failure may be explained by the down-regulation of
glucocorticoid receptors chronically exposed to elevated hormone levels

(Preisler et al., 2000a, 2000b).

25

Additionally, it has been reported that intramuscular lipids were
increased in rabbits that were infused with adrenocorticotropic hormone
(ACTH) (Romans et al., 1974) or dexamethasone (F redericks et. al.,
1975). In addition, there is some evidence suggesting that exogenous
glucocorticoid injected intramuscularly or subcutaneously can increase
intramuscular fat deposition in sheep (Harter and Vetter, 1967).

Fatty acids

Circulating fatty acids may have a role in both initiating adipose
differentiation and in initial filling of adipocytes. In cultured murine cell
lines, arachidonic acid (Czoz4) and cyclic AMP-elevating agents, especially
prostacyclin behave as mitogenic-adipogenic stimuli able to trigger
growth-arrested cells to enter the terminal phase of differentiation, and
thus appear to mimic the activity of glucocorticoids (Gaillard et al., 1991 ).
Arachidonic acid is found in only very low concentrations in common
feedstuffs. However, the essential fatty acid, linoleic acid (018:2), is a
precursor of arachidonic acid biosynthesis and is relatively high in several
oilseeds. One of the richest sources of linoleic acid is sunflower oil.
Although extensive biohydrogenation of unsaturated fatty acids occurs in
the reticulo-rumen, the degree of biohydrogenation is dependent upon
factors such as form of dietary fat, passage rate, and microbial population
(Allen, 2000). Ruminal biohydrogenation of sunflower oil fed to lactating

cows was approximately 80% (Kalscheur, et al., 1997) and that of

26

unsaturated 18C fatty acids in corn oil fed to beef steers was
approximately 70% (Kennington et al., 2000).

Hood (1983) demonstrated that appreciable rates of fatty acid
synthesis were occurring only in larger adipocytes in ovine adipose tissue,
especially in intramuscular adipose tissue. Seemingly, preadipocytes
need to be supplied with fatty acids for complex lipid biosynthesis, as
endogenous synthesis from glucose is still undetectable in contrast with
mature adipocytes (Ailhaud, 1996). Subsequently, fatty acids via fatty
acid-activated receptor (FAAR) and adipogenic hormones participate in
the formation of adipocytes from preadipocytes (Amri et al., 1994). Taken
together, these reports indicate that preadipocytes and “young” adipocytes
may be heavily dependent on the uptake of circulating fatty acids to
provide substrate for triacylglyceride biosynthesis. However, to sufficiently
establish a mode of action for these signals, it would be necessary to
establish changes on a molecular or gene expression level.
cDNA microarra y analysis

The bovine genome is composed of thousands of different genes
that are responsive to the environment of the animal. Recent
developments of cDNA microarray technologies offer the possibility of
analyzing the responsiveness of vast numbers of genes to external stimuli
(Yao et al., 2001 ). Although these technologies provide a means for
examining gene responsiveness to treatment, nutrition, disease, and

environment on a large scale, the resources to develop these technologies

27

for livestock species have been lacking (Yao et al., 2001). Recent
advances in the creation of tissue, cell, or species-specific cDNA libraries
for generation of thousands of expressed sequence tags (ESTs) make it
possible to examine differential gene expression in tissues such as
subcutaneous and intramuscular adipose. This is known as functional
genomics.

To date, a number of studies have utilized cDNA microarray
technology to examine differential expression in adipose tissue. Most of
this work has been confined to rodent species and has dealt with gene
expression differences between white adipocytes and brown adipocytes to
examine their unique metabolism (Reue and Glueck, 2001; Boeuf et al.,
2001; Boeuf et al., 2002).

Although functional genomics studies have been conducted with
cattle utilizing differential display-polymerase chain reaction (Childs et al.,
2002), the use of microarray analysis has been more limited due to lack of
availability of cDNA libraries specific to the bovine species. However, in
recent years the Center for Animal Functional Genomics at Michigan State
University has worked to develop cDNA libraries for domestic livestock
species. As a result of this work, a normalized cDNA library generated
from bovine total leukocytes (BOTL) and containing 842 unique cDNAs
has become available (Burton et al., 2001; Yao et al., 2001). Although
originally designed for studies in bovine immunobiology, most of the

differentiation and proliferation genes of leukocytes are ubiquitous in other

28

cells, expanding the usefulness of microarrays spotted with these cDNAs
to the study of multiple bovine cell types, including adipocytes.

Adipose tissue devel0pment in beef cattle comprises the topic of a
wide range of research within the animal industry. Although
glucocorticoids have been studied extensively in relation to adipose cells,

experiments with glucocorticoids and bovine adipose are limited.

. ”-1

Additionally, most studies that have been carried out in this area do not

fully explore the response of bovine adipose on a cellular or molecular

"\ It -

level.

Despite its possible uses in animal species, cDNA technology and
microarray analysis have seen only limited use in experiments with
production livestock. With cDNA microarray analysis representing a new
and powerful tool to examine gene expression and consequent phenotype
in cattle, it seems logical to apply this technology to an area that has
lacked conclusive research with conventional methods. Because
glucocorticoids appear to act differently in subcutaneous and
intramuscular depots, our hypothesis was that intramuscular and
subcutaneous adipose tissue would exhibit differential response to
dexamethasone treatment. Therefore, the objectives of these studies
were to characterize the response of subcutaneous and intramuscular
adipose in relation to glucocorticoids and to identify unique characteristics

of the two depots on a molecular level.

29

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38

CHAPTER II

INFLUENCE OF SUPPLEMENTAL SUNFLOWER OIL AND
DEXAMETHASONE THERAPY ON ADIPOSE TISSUE DEVELOPMENT
AND PERFORMANCE IN EARLY WEANED
BEEF STEERS

ABSTRACT

The purpose of this feedlot study was to investigate the effect of
dexamethasone therapy and supplemental sunflower oil on the
development of intramuscular adipose tissue in early weaned beef steers.
Twenty-four early weaned steers (182 i 21.7 kg) were randomly assigned
to a 2 x 2 factorial arrangement of either basal diet only (CON) or basal
diet with the addition of 8% high-linoleate sunflower oil (OIL) and
intramuscular injections every 28 d either of dexamethasone (0.1 mg
dexamethasone/kg body weight) (DEX) or an equal volume of
physiological saline (SAL) over a period of 112 d. Steers were fed
individually and amount fed was adjusted weekly so that treatment groups
had isocaloric intake. Following the 112 cl treatment period, all steers
were fed a common basal diet for 138 d and then harvested.
Accumulation of ultrasound measured intramuscular fat percentage,
subcutaneous rib fat and rump fat was not significantly effected by
treatment. Carcass marbling score, rib fat, and yield grade of treatment

groups were not significantly different than control. Additionally, proximate

39

analysis of rib steaks from carcasses showed no differences in
intramuscular lipid content and fatty acid analysis revealed few significant
differences in fatty acid profile between treatment groups. However, DEX
increased (P < 0.05) and OIL tended to increase (P = 0.07) kidney, pelvic,
and heart fat. Dexamethasone lowered ADG (P < 0.01) and feed
efficiency (P < 0.05) during the treatment period. However, during the
finishing period, DEX tended to increase ADG (P = 0.09) and feed
efficiency was increased by previous administration of DEX in combination
with OIL (P < 0.05). Furthermore, over the entire trial, an interaction
between DEX and OIL decreased feed efficiency (P < 0.05). In
conclusion, neither treatment was successful in influencing the
accumulation of adipose in intramuscular or subcutaneous depots.
Additionally, dexamethasone administration negatively affected feedlot
performance during the treatment period but increased performance

during the finishing period.

40

 

Introduction

Intramuscular fat, or marbling, is the major determinant of USDA
Quality Grade. Cattle are often fed for extended periods of time to
maximize marbling, because the sequence of adipose maturation from
ear1y to late is: perirenal, subcutaneous, interrnuscular and intramuscular
(Hood, 1983). Therefore, cattle often accumulate large amounts of
subcutaneous fat before marbling can reach desired levels. It is well
established that glucocorticoids, or the glucocorticoid analog
dexamethasone, enhance differentiation in the presence of insulin or
serum in nearly every preadipocyte cell line studied (Martin et al., 1998).
Even though adrenal glucocorticoids readily stimulate mobilization of lipid
from subcutaneous and other fatty depots, circulating levels of
endogenous glucocorticoids have been positively correlated with
intramuscular fat deposition in cattle (Cramer and Shahied, 1974; Romans
et al., 1974). In addition, there is limited evidence suggesting that
intramuscular or subcutaneous injections of exogenous glucocorticoid can
increase intramuscular fat deposition in cattle when administered late in
the feeding period (Brethour, 1972).

In studies with mouse preadipocyte cell lines, glucocorticoid effects
appear to be mediated through increased production of prostacyclins from
arachidonic acid (Ailhaud et al., 1992). Arachidonic acid is found in only

very low concentrations in common feedstuffs. However, linoleic acid

41

(C132) is a precursor of arachidonic acid (C20;4) biosynthesis and is in
relatively high concentration in several oilseeds. One of the richest
sources of linoleic acid is sunflower oil. Therefore, the objectives of this
study were to determine the influence of sunflower oil supplementation,
dexamethasone, and their combination, on adipose development and

performance in early weaned beef steers.

42

Materials and Methods

Twenty-four, spring-bom, Simmental x Angus half-sibling steer
calves were used to determine the effects of dexamethasone and
sunflower oil supplementation on intramuscular fat accretion. The dams of
these steers were maintained at the Michigan State University Upper
Peninsula Experiment Station, Chatham, Ml. Calves were weighed at
birth and castrated. Two wks before weaning, steers were vaccinated
against clostridial infections (UltraBac 7, Pfizer Inc., New York, NY) and
common respiratory diseases (CattleMaster 4 plus L5, Pfizer Inc.) Steers
were weaned at an average age of 110 d, weighed and transported 650
km to the Beef Cattle Teaching and Research Center, East Lansing, MI.
Calves were given a 50 d adjustment period while being stepped up to a
high concentrate diet. Calves were given booster vaccinations
(Bovashield 4, Pfizer, Inc. and Ultrabac 7) 14 d after arrival at the feedlot,
and were treated for internal and external parasites (lvomecPlus, Merial,
Whitehouse Station, NJ) 21 d after arrival at the feedlot and before the
initiation of treatments. Groups of six steers were housed in 13 x 3 m
partially-covered outdoor pens and were adapted to individual feeders
(American Calan lnc., Northwood, NH). None of the steers received
anabolic implants during their lifetime to avoid possible confounding

effects on intramuscular fat accretion.

43

Steers were allotted by age (average of 110 d) and weight (182 i
21.7 kg), and randomly assigned to a 2 x 2 factorial arrangement of
dietary and drug therapy treatments for a period of 112 d. Dietary
treatments (Table 2-1) were basal diet only (CON) or basal diet with the
addition of 8% high-Iinoleate, refined, bleached, and deodcrized sunflower
oil (OIL) (Columbus Foods Co., Chicago, IL). Fatty acid composition of
the sunflower oil is presented in Table 2-2. Calves were also assigned to
saline (SAL) or dexamethasone (DEX) treatment bouts every 28 d during
the treatment period. Following the 112-d treatment period, all steers
were fed a common basal diet until harvest at approximately 15 mo of
age.

Because the composition of diets during the treatment period was
not isocaloric, steers were organized into feeding groups to control calorie
intake. Steers were organized into groups of four by DMI during the
adjustment period so that each treatment group was represented once
within each feeding group. Orts were removed, sampled, and weighed
weekly, and fresh feed was offered daily to each steer. At the beginning
of each wk the steer with the lowest energy intake from the previous wk
was given ad libitum access to feed. The other three steers in the group
were fed to meet the ad libitum fed steer's energy intake from the previous
wk. The purpose of the feeding group system was to maintain isocaloric

intake among treatments. Steers were switched from a diet containing an

44

average of 12.8% CP to one containing 11.7% CP (Table 2-1) at the end
of the treatment period.

Injections of saline or dexamethasone were given at 0, 12, and 24 h
after the start of each 28-d period. Dexamethasone-treated calves
received intramuscular injections of 0.1 mg dexamethasone/kg body
weight (Azium®, Schering Corp, Kenilworth, NJ; 2 mg
dexamethasone/mL). Control calves received an equivalent volume of
physiological saline. Consecutive injections were given on alternating
sides of the neck.

Blood samples were collected by jugular venipuncture into 5-mL
evacuated tubes containing citrate-dextrose on d 0, 1, and 7 of each 28-d
period. Whole blood samples were used for immunostaining and flow
cytometric analysis of L-selectin on blood leukocytes as described by
(Burton et al., 1995). L-selectin is a neutrophil adhesion molecule that is
down-regulated in dexamethasone treated cattle (Burton and Kehrli,
1995). L-selectin level was used to verify the effectiveness of the
dexamethasone treatment and to monitor the return of normal immune
function in the calves before the following bout of injections was
administered.

Steers with rectal temperatures over 40°C (103.5°F) during the trial
were considered to be morbid and those with clinical signs of bacterial

infection were treated with Micotil (Elanco Animal Health, Indianapolis, IN),

45

Baytril (Bayer Animal Health, Shawnee Mission, KS), or Nuflor (Schering-
Plough Corp., Union, NJ) per label instructions.

Initial and final steer body weights were calculated by averaging
two weights taken on consecutive days. Interim weights and ultrasound
measures were taken every 28 d throughout the finishing period.
Percentage of lipid in the longissimus dorsi muscle, 12m-rib and rump
subcutaneous fat, and 12m-rib longissimus dorsi area was evaluated using
real-time ultrasound (Pie 200 SLC, Pie Medical, Tequesta, FL) by an
Animal Ultrasound Practicioners Assoc. certified technician.

Experimental procedures in this trial were conducted according to
those approved by the Michigan State University All University Committee
on Animal Use and Care (AUF No. 09/00-131-00).

Feedstuffs were sampled every 21 d. Samples for each of the
feedstuffs were ground through a Wiley mill fitted with a 1 mm screen
(Arthur H. Thomas, Philadelphia, PA). After grinding, samples were
combined into a single composite sample for each feedstuff. Dry matter
was determined by drying samples in a forced air oven for 24 h at 102°C.
Combustion method 990.03 (AOAC, 1995; Leco FP—2000, Leco Corp., St.
Joseph, MI) was used to determine CP. Total lipid was determined by
solvent extraction method 991.36 (AOAC, 1995; Tecator Soxtec System
HT 1046 service unit and Tecator Soxtec System HT 1043 extraction unit,
Tecator, lnc., Herndon, VA). Neutral detergent fiber was determined using

an ANKOM 200/220 Fiber Analyzer (ANKOM Technology. Macedon, NY).

46

Steers were harvested at Murco Foods, Plainwell, Ml. Carcass
data were collected 48 h postmortem. Carcass measurements collected
included hot carcass weight, longissimus muscle area, adjusted 12th rib
subcutaneous fat thickness, marbling score, USDA Quality Grade, and
skeletal maturity. Kidney, pelvic, and heart fat was trimmed from the
carcass and weighed.

An 11-12 rib section was removed from the right side of each
carcass and then frozen. The frozen rib section was fabricated into 2.5
cm thick steaks. Each steak was numbered sequentially starting from the
anterior end of the rib section. Moisture, CP, lipid (Steak #1), and fatty
acid composition (Steak #2) were determined in duplicate from powdered
samples. Powdered samples were prepared by denuding frozen steaks of
the spinalis dorsi muscle, external fat and connective tissue, so that only
the longissimus dorsi muscle remained. Then, the frozen longissimus
dorsi was sliced (0.5 cm thick) and cut into small pieces. Pieces of
chopped muscle, along with dry ice, were powdered using a Tecmar
grinder (Tecmar Co., Cincinnati, OH). Dry ice was allowed to evaporate
before powdered samples were stored at -20°C. Moisture was determined
by drying powdered muscle samples in a forced air oven for 18 h at 102°C
(method 950.46; AOAC, 1995). Combustion method 992.15 (AOAC,
1995; Leco FP-2000, Leco Corp., St. Joseph, MI) was used to determine

crude protein. Total lipid was determined as described for feed samples.

47

Dietary ingredients as well as the longissimus muscle (Steak #2)
from each steer were homogenized and used for FA analysis. Samples
were prepared for FA analysis as methyl esters (Sukhija and Palmquist,
1988) with nervonic acid (024:1) as the internal standard. Concentrations
of FA were determined by using a gas chromatograph (Varian 3800,
Varian Chromatography Systems, Wallnut Creek, CA) on a fused silica
capillary column (100 m x 0.32 mm [SP 2560]; Supelco, lnc., Bellefonte,
PA). The separation was achieved by a temperature gradient program
beginning at 50°C and increased to 200°C at 15°C/min, held at 200°C for
25 min, raised to 225°C at 1°C/min, and finally held at 225°C for 27 min.
.The temperatures of the injection port and the flame ionization detector
were set at 180 and 230°C, respectively. Helium was used as the carrier
gas (flow rate of 1.5 mL/min with a total column pressure of 25 psi) with a
split ratio of 100:1. Peaks were identified by comparisons with authentic
standards and quantified by peak area integration.

Statistical analyses. Performance and tissue composition data
were analyzed using the General Linear Model procedure of SAS (SAS
Inst. Inc., Cary, NC) with individual steer as the experimental unit. The
model included a random effect of steer, and fixed effects of diet,
dexamethasone-treatment, and diet x dexamethasone-treatment
interaction as independent variables. Data variables collected over time
such as weight and ultrasound measures were analyzed using the

appropriate repeated measures procedures. Two steers, both from the

48

dexamethasone-treated group, died during the course of the 112 d
treatment period. Neither of the steers were observed to be morbid before
death and no steers in the DEX group showed clinical signs of morbidity
during the trial. Data from these two steers were not included in the
analysis. The level of probability at which main effects and interactions

were considered significant was P < 0.05 and P < 0.10, respectively.

'u'. .r.’

49

Results and Discussion

Feedlot performance. Dexamethasone resulted in a decrease in
DMI (P < 0.05), ADG (P < 0.01 ), and feed efficiency (P < 0.05) during the
112 d treatment period (Table 2-3). The decreased performance during
this period of the trial may have been the result of a down-regulation of the
immune system due to the acute dexamethasone treatment bouts.
Results from blood samples of steers confirmed a down regulation of
neutrophil adhesion molecules in DEX treated steers (Weber et al., 2001).
Dexamethasone has been shown to down regulate key immune regulatory
molecules on neutrophils (Burton et al., 1995). This results in decreased
white blood cells being targeted to infected tissues and a consequent
decrease in immune response. During the trial steers treated with DEX
were observed to have more instances of fever that required treatment
with antibiotics. Steers that received DEX were more likely to need one
antibiotic treatment (P < 0.05) and were also more likely to need two or
more treatments (P < 0.01). Thus, steers receiving DEX appear to have
been fighting subclinical infections, decreasing ADG and feed efficiency.

The Mcal of NEg consumed during the treatment period as well as
DMI were not different between treatments, indicating that the strategy of
maintaining isocaloric intake between dietary treatment groups was

successful.

50

During d 113 to 251, DEX tended to increase ADG (P = 0.09) and
increased feed efficiency (P < 0.05). The increases in ADG and feed
efficiency during d 113 to 251 of the finishing period for the DEX group
may be attributed to compensatory gain as steers in the DEX group were
lower performing in gain and efficiency during the 112 d treatment period.
The increased feed efficiency during the 113 to 251 d finishing period was
diminished however, in combination with OIL (P < 0.05). Over the entire
trial (0 to 251 d), DEX appeared to increase feed efficiency, but increases
in feed efficiency were reduced by an interaction between DEX and OIL.
Because high lipid content of feed may serve to decrease ruminal
digestibility (Doreau and Chilliard, 1997), the decreases in feed efficiency
that were caused by OIL may be attributed to decreased digestibility of the
diet.

Ultrasound measures are presented in Table 2-4. Subcutaneous
fat thickness in DEX treated steers remained lower for the entire trial, even
after DEX treatment was terminated, however it was not a significant
decrease in accumulation. Overall DEX did not significantly affect
intramuscular lipid content. This agrees with the work of Romans et al.
(1974) in which it was observed that increases in exercise stress caused
lipolysis and increased circulating free fatty acids without detrimental
effects on intramuscular lipid deposition. lf subcutaneous tissue contains
a larger percentage of mature adipocytes per gram of tissue than

intramuscular adipose in cattle as observed by May et al. (1994), the

51

subcutaneous depot may experience a more acute reaction to the lipolytic
effect of glucocorticoids than intramuscular tissue, however in this study
not significant differences were seen in either depot. The failure of
dexamethasone to increase intramuscular lipid, however, is in
contradiction of the results of Brethour (1972). Brethour (1972) reported
that injections of exogenous glucocorticoids significantly raised
intramuscular fat content and marbling scores in steers that were
administered dexamethasone before harvest.

Differences In intramuscular fat content for the treatment groups as
determined by ultrasound every 28 d are plotted in (Figure 2-1).
Supplemental sunflower oil increased ultrasound intramuscular fat over
time compared to control but not to a significant degree. If the increased
availability of linoleic acid from sunflower oil supplementation resulted in
increased arachadonic acid synthesis and metabolism, a subsequent
increase in prostacyclins and CAMP-elevating agents may have resulted.
Aubert et al. (2000) concluded that similar conditions could result in an
adipogenic response. It is also possible that increases in intramuscular
lipid may have been elicited by the availability of readily esterifiable fatty
acids provided by the sunflower oil.

Dexamethsone may have exerted opposite effects on marbling at
different points during the trial. The apparent increase in intramuscular
lipid caused by DEX after the first treatment may have had the effect of

causing differentiation of preadipocytes thus maturing adipose cells in the

52

intramuscular tissue. With the second treatment, DEX caused a decrease
in intramuscular lipid. DEX may have exerted an opposite effect on the
mature cells, causing lipolysis and —a subsequent decrease in
intramuscular adipose tissue with later injections. Another explanation for
DEX failing to increase intramuscular fat over the entire trial period may be
due to the large, acute exposure to glucocorticoids resulting in a down-
regulation of glucocorticoid receptors and a decreased response (Preisler
et al., 2000). Corah et al. (1995) reported that dexamethasone did not
result in an increase in intramuscular lipid but only increased the external
fat depth of steers. The method of administration for dexamethasone in
the Corah et al., (1995) study was a long lasting implant instead of acute
injections, which may have resulted in a similar down-regulation of the
glucocorticoid receptor.

Differences in subcutaneous 12th rib backfat and subcutaneous
rump fat are plotted in Figure 2-2 and Figure 2-3, respectively.
Subcutaneous fat thickness did not show the same erratic increases and
decreases in response to DEX treatments that were observed in
intramuscular adipose.

Carcass marbling score and overall USDA Quality Grade of
treatment groups were not significantly different from control (Table 2-5),
and there were not significant differences in lipid content of the
longissumus dorsi muscle of steers measured by proximate analysis

(Table 2-6). Although ultrasound rib and rump fat were decreased by DEX

53

over the entire trial period, there were no significant differences in 12th rib
fat thickness of carcasses or final yield grade. However, kidney, pelvic,
and heart fat percentage was increased by DEX (P < 0.05) and tended (P
= 0.07) to be increased by OIL.

Results of the fatty acid (FA) analysis on the longissimus dorsi
muscle from the rib steaks of steers are presented in Table 2-7.
Percentages of individual FA as compared to total FA were generally not
significantly different and different treatment groups exhibited similar
profiles.

Conjugated linoleic acid (CLA), a product of incomplete microbial
biohydrogenation of linoleic acid did not significantly increase with the
supplementation of the high-linoleate sunflower oil. This disagrees with
previous work (Kennington et al, 2000; Mir et al., 2002) in which significant
increases in CLA formation were realized due to increased linoleic acid in
the diets of cattle. However, the 10,12 isomer of CLA, which is the less
abundant isoform, was increased in a highly significant fashion by OIL (P
< 0.01). The failure of sunflower oil to increase CLA content in the
intramuscular lipid of steers may be due to the extended finishing period
(138 d) that followed the end of sunflower oil supplementation. Because
ruminant FA composition is affected by dietary FA composition (Doreau
and Chilliard, 1997), the profile of intramuscular lipid may have been
altered over the finishing period. This may partially explain the differing

CLA content from this trial as compared to previous work.

Implications

Intramuscular and subcutaneous adipose tissue may be subject to
manipulation by glucocorticoids. Stimulation of intramuscular adipose
tissue by dexamethasone may be able to increase marbling at a particular
time or stage of tissue development. However, additional investigations
would be required to discover the optimum time and dosage of
dexamethasone administration to exert these effects. Additionally,
dexamethasone may be a useful tool in reducing subcutaneous adipose
fissue.

Supplementation of sunflower oil increased ultrasound measured
intramuscular lipid content in beef cattle, but not to a significant degree
and did not increase carcass marbling score. Furthermore, sunflower oil
supplementation did not significantly change fatty acid profile in rib steaks
or increase levels of CLA. As this is in contradiction with previously cited
work, it is most likely the effect of an extended finishing period with the
absence of sunflower oil. This suggests that the effects of sunflower oil on

adipose may only be realized when the oil is supplemented to harvest.

55

 

Table 2-1. Composition of finishinldiets (DM basis)
Diet d 0-112 Diet Cl 113-251

 

 

 

 

 

Ingredient CON OIL
-% of diet -

High moisture shelled corn 65.7 56.0 74.0
Corn silage 15.0 15.0 15.0
Soybean meal 11.9 14.2 5.8
Sunflower oil 0.0 8.0 0.0
Protein-mineral-vitamin supplementa 5.0 5.0 5.0
Calcium carbonate 0.52 0.52 0.0
Magnesium oxide 0.30 0.30 0.0
Components

Protein 13.2 12.3 11.7
NDF 15.2 13.3 15.4
Lipid 1.2 9.0 1.3
NEg, Mcal/kg 1.39 1.56 1.41

 

aProtein-mineral-vitamin supplement composition - soybean meal, 47.7%;
calcium carbonate, 22.1%; urea, 7.5%; potassium chloride, 5.6%;
dicalcium phosphate, 2.2%; ground corn, 3.3%; selenium 90 (198 mg/kg)
1.06%; Vitamin A (30,000 lU/g), 0.16%; Rumensin 80 (176 g
monensin/kg), 0.28%; Trace mineral salt (NaCl, 94%; Zn, 3500 ppm; Fe,
2000 ppm; Cu, 300 ppm; l, 70 ppm; Co, 50 ppm), 10.0%.

56

Table 2-2. Fatty acid profile of sunflower oil provided to steers
during the 112 d treatment period

 

 

Fatty acid % of Sunflower oil
16:0 palmitic acid 5.8

16:1 palmitoleic acid 0.1

18:0 stearic acid 3.0

18:1 oleic acid 0.4

18: 1 w—9 oleic acid 18.4
18:1w-7 oleic acid 0.7
18:2w-6 linoleic acid 68.4
18:3w-3 linolenic acid 0.8

Other 2.4

 

57

ImQ—i

Table 2-3. Finishing performance of steers that received saline (SAL) or
dexamethasone (DEX) treatments and received diets containing no
supplemental oil (CON) or sunflower oil QIL) for a 112-d treatment period

 

 

 

 

 

SAL DEX Prob.
CON OIL CON OIL SEMa DEX OIL DEXxOIL

No. of steers 6 6 5 5
DMI, kg/d

d 0-112 6.2 5.5 5.3 5.6 0.29 0.17 0.38 0.04

(I 113-251 7.9 7.8 7.0 7.8 0.41 0.34 0.39 0.28

d 0-251 7.1 6.7 6.3 6.8 0.27 0.22 0.73 0.11
NEg Intake,
Meal/db

d 0-112 8.66 7.68 7.51 7.94 0.33 0.17 0.38 0.04
d 113-251 11.3 11.1 10.1 11.20 0.58 0.34 0.39 0.27
d 0-251 10.1 9.54 8.90 9.70 0.41 0.23 0.72 0.11

ADG, kg/d

d 0-112 1.24 1.09 0.98 1.00 0.06 0.01 0.26 0.16
(I 113-251 1.29 1.31 1.47 1.32 0.06 0.09 0.24 0.13
d 0-251 1.27 1.22 1.27 1.19 0.05 0.76 0.13 0.71

Gain/feed
d 0-112 0.20 0.20 0.18 0.18 0.01 0.02 0.54 0.84
(I 113-251 0.19 0.19 0.25 0.19 0.01 0.02 0.09 0.03
d 0-251 0.19 0.20 0.22 0.19 0.01 0.22 0.06 0.03

 

8'Standard error of the mean.
bBased on energy value of feedstuffs according to 1996 NRC.

58

— Tin-i.

Table 2-4. Ultrasound measures of steers that received saline (SAL) or
dexamethasone (DEX) treatments and received diets containing no
supplemental oil (CON) or sunflower oil (OIL) for a 112 d treatment period

 

 

 

 

 

SAL DEX Prob.

CON OIL CON OIL SEMa DEX OIL DEXxOlL
No. of steers 6 6 5 5
Increase in
intramuscular
fat, % lipid
0-112 d 0.72 0.70 0.37 0.57 0.24 0.32 0.70 0.62
113-251 d 0.83 0.68 0.56 1.23 0.36 0.69 0.45 0.24
0-251 d 1.55 1.38 0.92 1.81 0.44 0.81 0.41 0.22
Increase in
12th rib fat
thickness, cm
0-112 d 0.20 0.15 0.15 0.20 0.043 0.96 0.77 0.32
113-251 d 0.43 0.46 0.36 0.36 0.071 0.22 0.80 0.86
0-251 d 0.63 0.61 0.51 0.56 0.101 0.38 0.96 0.76
Increase in
rump fat
thickness, cm
0-112 d 0.23 0.13 0.12 0.13 0.056 0.33 0.31 0.23
113-251 d 0.31 0.38 0.22 0.25 0.048 0.02 0.24 0.59
0-251 d 0.55 0.51 0.33 0.38 0.074 0.03 0.99 0.56

 

aStandard error of the mean.

59

 

Table 2-5. Carcass data for steers that received saline (SAL) or

dexamethasone (DEX) treatments and received diets containing no

supplemental oil (CON) or sunflower oil (OIL) for a 112 d treatment period
SAL DEX Prob.

 

 

 

CON OIL CON OIL SEMa DEX OIL DEXxOlL
No.ofsteers 6 6 5 5

Hot carcass 333 333 336 334 10.9 0.86 0.94 0.90
wt., kg

KPH fat, "/0 1.63 2.16 2.19 2.76 0.29 0.05 0.07 0.93

Longissimus 88.4 88.4 87.7 80.7 3.9 0.28 0.34 0.34
muscle area,
cmz

Adj.12"‘ribfat 1.24 1.19 1.12 1.30 0.20 0.96 0.78 0.55
thickness,cm

Yield grade 2.47 2.52 2.51 3.11 0.34 0.35 0.33 0.40
Marbling score” 450 393 398 448 26.8 0.96 0.90 0.05

Qualitygrade Ch- Se+ Se+ Ch-
aStandard error of the mean.
b300=Slight°, 400=Small°.

60

 

Table 2-6. Proximate analysis of longissimus dorsi muscle from rib steaks
of steers that received saline (SAL) or dexamethasone (DEX) treatments
and received diets containing no supplemental oil (CON) or sunflower oil
(OIL) for a 112 d treatment period

 

 

 

 

SAL DEX Prob.
CON OIL CON OIL SEMa DEX OIL DEXxOlL
No. of steers 6 6 5 5
Moisture,% 69.77 68.77 71.36 71.48 1.58 0.17 0.77 0.71
Intramuscular 5.39 4.32 3.64 4.46 0.61 0.19 0.83 0.12
lipid,%
Protein, % 30.74 29.36 26.10 27.83 2.10 0.14 0.93 0.45

 

8'Standard error of the mean.

61

 

Table 2-7. Fatty acids as a percentage of total fatty acids for steers that
received saline (SAL) or dexamethasone (DEX) treatments and received
diets containing no supplemental oil (CON) or sunflower oil (OIL) for a 112
d treatment period

 

 

 

 

 

SAL DEX Prob.

CON OIL CON OIL SEMa DEX OIL DEXxOlL
No. of steers 6 5 5 5
Myristic acid 2.930 2.647 2.829 3.039 0.239 0.54 0.88 0.31
(14:0)
Myristoleic 0.043 0.369 0.502 0.090 0.255 0.72 0.87 0.16
acid (14:1)
Palmitic acid 27.57 26.87 27.50 27.36 0.566 0.70 0.45 0.62
(16:0)
Palmitoleic 3.552 2.783 3.210 3.352 0.172 0.51 0.08 0.02
acid (16:1)
Stearic acid 12.41 14.33 13.25 12.78 0.470 0.45 0.14 0.02
(18:0)
Oleic acid
(18:1 n9)
-cis-isomer 40.81 38.27 39.67 39.52 1 .370 0.96 0.24 0.30
-trans isomer 2.372 3.610 2.293 3.603 0.337 0.90 <0.01 0.91
Linoleic acid
(18:2n6)
-cis-isomer 3.329 4.305 3.745 3.552 0.340 0.62 0.26 0.10
-trans-isomer 0.059 0.051 0.049 0.050 0.011 0.62 0.81 0.67
Cojugated
linoleic acid
-9,11 isomer 0.236 0.200 0.224 0.283 0.026 0.19 0.65 0.08
-10,12 isomer 0.007 0.047 0.002 0.037 0.011 0.52 <0.01 0.84
Arachadonic 0.001 0.004 0.003 0.003 0.001 0.72 0.56 0.46
acid (20:4n9)

 

aStandard error of the mean.

62

Figure 2-1. Ultrasound readings of intramuscular fat taken at 28 d
increments for steers that were control, received supplemental sunflower
oil in the diet (OIL), received treatments of dexamethasone (DEX), or
received supplemental sunflower oil in the diet and received treatments of
dexamethasone (OIL+DEX).

i—e—Control - .D . OIL —. -DEX —o— OIL + DEX \

 

 

 

 

 

 

1.75

 

1.5 -

1.25 -

 

 

0.75

 

 

 

0.5

Change in longissimuss IMF, %

0.25 .

 

 

 

 

 

 

-0.25

 

0 28 56 84 112 140 168 196 224 252
Day oftrial

63

Figure 2-2. Ultrasound readings of subcutaneous 12th rib fat taken at 28 d
increments for steers that were control, received supplemental sunflower
oil in the diet (OIL), received treatments of dexamethasone (DEX), or
received supplemental sunflower oil in the diet and received treatments of
dexamethasone (OIL+DEX).

I—O—Control - EI- OIL —-A-oEx —-o- clL+oExi

 

 

 

 

 

0.7
,o
06 i — fiv———— .1.
I F7
05 + f- — .—- ———‘n ‘
,EI ,/
,

P
k
\

 

l
l

 

 

.0
N

Change in 12th Rib Fat Depth, cm.

.0
—L

 

 

 

 

O

 

0 28 56 84 112 140 168 196 224 252
Day of trial

64

Figure 2-3. Ultrasound readings of subcutaneous rump fat taken at 28 d
increments for steers that were control, received supplemental sunflower
oil in the diet (OIL), received treatments of dexamethasone (DEX), or
received supplemental sunflower oil in the diet and received treatments of
dexamethasone (OIL+DEX).

 

 

Le—Control - El - out —1A—DEX —o- clung

|
L. __

 

 

 

0.6

 

 

 

 

 

 

 

 

Change in Rump Fat Depth, cm.

 

 

 

 

0 28 56 84 1 12 140 168 196 224
Day of Trial

65

 

 

Literature Cited

Ailhaud, G., P. Grimaldi, and R. Negrel. 1992. Cellular and molecular
aspects of adipose tissue development. Annu. Rev. Nutr. 12:207-
233.

Aubert J., P. Saint-Marc, N. Belmonte, C. Dani, R. Negrel, and G. Ailhaud.
2000. Prostacyclin lP receptor up-regulates the early expression of
C/EBPbeta and C/EBPdeIta in preadipose cells. Mol. Cell.
Endocrinol. 160:149-156.

AOAC. 1995. Official Methods of Analysis (16th Ed.) Association of
Official Analytical Chemists, lnc., Arlington, VA.

Brethour, J. R. 1972. Effects of acute injections of dexamethasone on
selective deposition of bovine intramuscular fat. J. Anim. Sci.
35:351-356.

Burton, J. L., and M. E. Kehrli. 1995. Regulation of neutrophil adhesion
molecules and shedding of Staphylococcus aureus in milk of

cortisol- and dexamethasone-treated cows. Am. J. Vet. Res.
56:997-1006.

Burton, J. L., M. E. Kehrli, S. Kapil, and R. L. Horst. 1995. Regulation of L-
selectin and CD18 on bovine neutrophils by glucocoticoids: effects
of cortisol and dexamethasone. J. Leukocyte Biol. 57:317-325.

Corah, T. J., J. D. Tatum, J. B. Morgan, R. G. Mortimer, and G. C. Smith.
1995. Effects of a dexamethasone implant on deposition of

intramuscular fat in genetically identical cattle. J. Anim. Sci.
73:3310-3316.

Cramer, D. A., and l. l. Shahied. 1974. Plasma corticoids and fat
deposition in the bovine. Proc. West. Sec. Amer. Soc. Anim. Sci.
25:108. (Abstr.).

Doreau, M., and Y. Chilliard. 1997. Digestion and metabolism of dietary fat
in farm animals. Br. J. Nutr. 78:815-835.

Hood, R. L. 1983. Changes in fatty acid synthesis associated with growing
and fattening. Proc. Nutr. Soc. 42:303-313.

66

Kennington, L. R., S. K. Duckett, J. G. Andrae, C. W. Hunt, F. N. Owens,
and G. T. Pritchard. 2000. Effect of high oil corn or added corn oil
on ruminal biohydrogenation and conjugated linoleic acid formation.
J. Anim. Sci. 78(Suppl. 1.):276. (Abstr.).

Martin, R. J., G. J. Hausman, and D. B. Hausman. 1998. Regulation of
adipose cell development in utero. Proc. Soc. Exp. Biol. Med.
219:200-210.

May, S. G., J. W. Savell, D. K. Lunt, J. J. Wilson, J. C. Laurenz, and S. B.
Smith. 1994. Evidence for preadipocyte proliferation during culture
of subcutaneous and intramuscular adipose tissues from Angus
and Wagyu crossbred steers. J. Anim. Sci. 72:3110-3117.

Mir, P. 8., Z. Mir, P. S. Kuber, C. T. Gaskins, E. L. Martin, M. V. Dodson,
J. A. Elias-Calles, K. A. Johnson, J. R. Busboom, A. J. Wood, G. J.
Pittenger, and J. J. Reeves. 2002. Growth, carcass characteristics,
muscle conjugated linoleic acid (CLA) content and response to
intravenous glucose challenge in high percentage Wagyu, Wagyu x
Limousin, and Limousin steers fed sunflower oil-containing diets. J.
Anim. Sci. 80:2996-3004.

Preisler, M. T., P. S. D. Weber, R. J. Tempelman, R. J. Erskine, H. Hunt,
and J. L. Burton. 2000. Glucocorticoid receptor down-regulation in
neutrophils of periparturient cows. Am. J. Vet. Res. 61:14-19.

Romans, J. R., H. W. Norton, l. S. Palmer, D. R. Wenger, W. J. Costello,
H. J. Tuma, R. Ball, and R. C. Wahlstrom. 1974. Preslaughter
treatment affecting intramuscular and plasma lipids. |l. Effects of
marketing, fasting, and exercise in beef steers. J. Anim. Sci. 38:38-
46.

Sukhija, P. S., and D. L. Palmquist. 1988. Rapid method for determination
of total fatty acid content and composition of feedstuffs and feces.
J. Agric. Food Chem. 36:1202-1206.

Weber, P. S. D., S. A. Madsen, G. W. Smith, J. J. Ireland, and J. L.
Burton. 2001. Pre-translational regulation of neutrophil CD62L in

glucocorticoid-challenged cattle. Vet. Immunol. lmmunopathol.
83:213-240.

67

CHAPTER III
DIFFERENTIAL GENE EXPRESSION OF SUBCUTANEOUS AND

INTRAMUSCULAR ADIPOSE TISSUE FROM BEEF STEERS
TREATED WITH DEXAMETHASONE

ABSTRACT

The objective of this study was to investigate the effects of
dexamethasone on subcutaneous and intramuscular adipose tissue based
on differences in expressed genes. Four Angus steers (585 i 27 kg) of
similar origin were assigned to two groups and received either a single
injection of saline (CON) or of 0.1 mg dexamethasone/kg of body weight
(DEX) 24 h before harvest. Tissue samples of subcutaneous and
intramuscular adipose were obtained postmortem and immediately flash-
frozen in liquid nitrogen for subsequent RNA isolations. Analysis of
differential gene expression was carried out by using the isolated RNA to
probe bovine cDNA microarrays. The presence of glucocorticoid receptor
mRNA in each tissue was also examined using Northern blot
hybridization. The microarray analysis revealed that DEX up-regulated (P
< 0.01) expression of urokinase receptor mRNA in both adipose tissue
depots, suggesting induction of the mitotic cell cycle and preadipose cell
proliferation. In addition, intramuscular adipose tissue from both treatment
groups exhibited significantly greater expression of genes involved in
cellular proliferation (MDM2; c-myc; hypoxanthine

phosphoribosyltransferase; alpha-tubulin) than tissue collected from the

68

subcutaneous depot. Intramuscular adipose tissue was also observed to
have greater expression (P < 0.05) of four genes involved in cellular
apoptosis (Caspase—4, serine protease inhibitor p19, bax-alpha, and
TRAF5) than in subcutaneous tissue. Northern blot analysis confirmed
expression of the glucocorticoid receptor (GR) in both types of adipose
tissue; however, expression was significantly lower (P < 0.01) in
intramuscular than in subcutaneous adipose depots. Administration of
DEX lowered GR mRNA abundance in both adipose depots (P < 0.05),
suggesting negative feedback of the steroid on expression of its receptor.
The results of this study suggest that subcutaneous adipose tissue is
different from intramuscular adipose tissue at the level of expressed
genes, and that subcutaneous adipose depots may be more sensitive to
the lipolytic effects of glucocorticoids due to greater abundance of GR
than intramuscular depots. Further studies are needed for
characterization of the differences between the two depots to determine

the reasons behind the differential gene expression that is observed.

69

Introduction

Evidence has accumulated to indicate that intramuscular
adipocytes represent a cell population different from more extensively
investigated subcutaneous adipocytes (Lin et al., 1992). In examining the
differences between adipose tissue depots on a cellular level it was
observed that intramuscular adipocytes had a smaller cell diameter and
cell volume than subcutaneous adipocytes (May et al.. 1994). Adipocytes
from subcutaneous depots are larger and more spherical in shape and
contain greater accumulation of triacylglycerides than adipocytes from
intramuscular depots. This is likely due, in part, to a greater number of
adipocytes being more mature at a given age of the animal in
subcutaneous versus intramuscular fat. Terminal cellular differentiation is
a process in which cells begin to arrest the proliferative cycle and take on
characteristics of a specific mature cell type. The greater accumulation of
triacylglycerides exhibited in subcutaneous adipocytes may be the result
of a greater number of differentiated adipocytes in that depot.
Glucocorticoid, or its synthetic analog dexamethasone, is routinely used in
adipocyte cell cultures to induce adipocyte differentiation (Martin et al.,
1998).

Because of the apparent biological differences of adipocytes
among depot locations and the influence of glucocorticoid hormones on

adipocyte differentiation, the purpose of this study was to identify unique

70

aspects of intramuscular and subcutaneous adipose on a molecular level
as well as to characterize a response to dexamethasone treatment within

the two depots by surveying differences in expressed gene profiles.

71

Materials and Methods

Four Angus steers of similar origin (585 i 27 kg) were allotted by
ultrasound intramuscular fat percentage to one of two treatments. Steers
received either an injection of 0.1 mg dexamethasone/kg body weight
(DEX) (Azium®, Schering Corp, Kenilworth, NJ; 2 mg dexamethasone/mL)
or an equivalent volume of physiological saline (CON). A single injection
was administered intramuscularly in the neck 24 h before harvest.

Sample collection. Steers were harvested at the Michigan State
University Meats Laboratory. Boneless rib steaks approximately 5 to 8 cm
thick were obtained from between the 10th and 12th rib immediately after
exsanguination by cutting directly through the hide. Sections of
subcutaneous fat were sliced into approximately one gram pieces. The
portion of longissimus dorsi muscle was dissected and tissue snips of
intramuscular adipose tissue were removed. Care was taken to obtain
sample free from visible muscle fibers. The tissue snips were snap frozen
in liquid nitrogen, collected into 50 mL conical polypropylene tubes and
placed on dry ice. Each of the two sample types (subcutaneous and
intramuscular adipose tissue) was collected within 30 min of
exsanguination. Upon the completion of sample collections, samples
were stored at -80°C.

Extraction of total RNA. Total RNA was extracted using TRlzol®

reagent (Life Technologies, Gaithersburg, MD) by the following

72

procedures. Tissue samples were homogenized in 1 mL of TRlzol®
reagent per 50 to 100 mg of tissue. Homogenized samples were
incubated for 5 min at 30°C after which 0.2 mL of chloroform was added
per 1 mL of TRlzol®. The sample was mixed, incubated for a second time
at 30°C for 3 min and centrifuged at 12,000 x g for 10 min. Following
centrifugation, the colorless, aqueous phase containing RNA was
removed and the RNA precipitated using isopropyl alcohol at 0.5 mL per 1
mL of TRlzol®. Samples were then incubated at 30°C for 10 min and
centrifuged at 12,000 x g for 10 min. After the RNA was precipitated, it
was washed with 75% ethanol using 1 mL of 75% ethanol per 1mL of
original TRlzol® reagent and centrifuged at 12,000 x g for 10 min. The
75% ethanol wash was then repeated. After extraction, RNA was dried
and redissolved in RNase—free water by passing the solution through a
pipette and incubating for 10 min at 55°C. One-tenth the sample volume
of 3 M sodium acetate and 2.5 times the sample volume of 100% ethanol
were then added and mixed with sample for precipitation. The dissolved
sample was then stored at -80°C for overnight precipitation of RNA.
Samples were then recovered and dissolved in RNAse-free water.
Concentration and purity of RNA was determined by analyzing a 1/10
dilution of each sample using a DU-650 spectrophotometer (Beckman,
Schaumberg, IL). After determination of concentration, integrity of RNA

samples was checked by ethidium bromide staining and visualization of

73

gel electrophoresed 28S and 188 rRNA bands using procedures outlined
by Weber et al. (2001).

RNA analysis. Microarray analysis of differential gene expression
among adipose tissue samples was carried out in the Michigan State
University Center for Animal Functional Genomics.

Samples of RNA from the same location and treatment group were
pooled for analysis to obtain the necessary 20 pg for microarray analysis.
Microarray analysis was performed in a loop design (Kerr and Churchill,
2001) to examine differences between subcutaneous and intramuscular
adipose tissue samples with and without dexamethasone treatment. The
design included four microarray slides that provided for each sample to be
labeled with both Cy3 (green) and Cy5 (red) fluorescent dyes (Amersham
Pharmacia Biotech. Piscataway, NJ) once during the design. The four
arrays that comprised the design were prepared to demonstrate four
different comparisons of adipose tissue sample: (1) subcutaneous,
dexamethasone-treated (Cy5) versus subcutaneous, control (Cy3) (2)
intramuscular, dexamethasone-treated (Cy3) versus intramuscular, control
(Cy5) (3) subcutaneous, dexamethasone-treated (Cy3) and intramuscular,
dexamethasone-treated (Cy5) (4) subcutaneous, control (Cy5) versus
intramuscular,control (Cy3).

Synthesis of cDNA was carried out using the Atlas Glass
Fluorescent Labeling Kit (Clonetech Laboratories, Palo Alto, CA).

Fluorescent dye labeling and cDNA purification were carried out according

74

to the manufacturer’s instructions. Samples were then used to interrogate
cDNA microarrays containing 1056 unique bovine ESTs derived from a
bovine total leukocyte library (Burton et al., 2001; Yao et al., 2001) and
targeted genes involved in transcription, cell signaling, cell migration, and
apoptosis (Coussens and Nobis, 2002). Probe was allowed to hybridize to
a microarray slide for 18 h using a GeneTAC Hybridization Station
(Genomic Solutions, Ann Arbor, MI). The hybridized slide was scanned
using a GeneTAC LS 4 and GeneTAC Integrator 3.3 software (Genomic
Solutions, Ann Arbor, MI) was used to obtain spot ratios from the scanned
microarray slide.

Northern hybridization. Northern blot analysis was carried out
using procedures outlined by Weber et al. (2001). Samples of RNA for
Northern blot analysis of GR mRNA abundance were pooled as described
above for microarray analysis, providing four different treatment groups.
Each of the four samples were loaded in duplicate across a total of eight
lanes (10 pg per lane) and electrophoresed in a 1.2% agarose denaturing
gel. Separated RNA was then transferred to a nylon membrane by use of
a Turbo Blotter apparatus (Schleicher and Schuell Bioscience, Inc. USA,
Keene, NH) according to the manufacturer’s instructions. RNA was UV
crosslinked on the membrane. The membrane was then pre-hybridized at
42°C for 4 h with a solution containing 50% formamide, 5X Denhardt’s, BX
880, 0.1% 808, 0.05 M phospate buffer, 1.0 mM EDTA, and 0.15 mg per

mL yeast tRNA. Samples of mRNA were hybridized in the same solution

75

at the same temperature for 18 h with a 32P labeled probe for the
glucocorticoid receptor 9-a subunit. The GR cDNA probe used in this
study was obtained from Dr. J. L. Burton (Michigan State University, East
Lansing, MI). The cDNA was developed by PCR amplification using a
primer pair designed from sequence of human GR exon 9-a (Hollenberg
et al., 1985; Encio and Detera-Wadleigh, 1991) and Holstein genomic
DNA as the template. The 1281 bp PCR amplification product was cloned

and sequenced as described by Weber et al., (2001) and was 88%

 

homologous to corresponding cDNA sequence of exon 9-a in the human
GR cDNA (Hollenberg et al., 1985). Following probe hybridization, the
membrane was washed at room temperature with 2X SSC, 0.1% SDS for
15 min and three times with 0.1X SSC, 0.1% SDS at 65°C. The
membrane was then exposed to BioMax MS film (Fisher Scientific,
Pittsburgh, PA) for 8 d at -80°C. The membrane was then stripped of
radioactive probe and rehybridized as described above with a 32P labeled
probe for B—actin. The B-actin cDNA was obtained from Dr. L. Kedes
(Stanford University School of Medicine; Palto Alto, CA) and is described
by Ponte et al. (1984) and donated for this study by Dr. J. L. Burton. The
membrane was exposed to film at -80°C for 2 d. Quantitative analysis of
mRNA abundance was measured using a 63-710 calibrated imaging
densitometer and multi-analyst software (Bio-Rad Laboratories, Hercules,

CA) and GR expression was shown as a ratio to beta actin expression.

76

Statistical analysis. Data from each array were normalized using
the LOESS procedure of SAS (SAS Inst. Inc., Cary, NC) as described by
Cleveland and Grosse (1991). Normalization was assessed by
constructing M—A scatter plots before and after normalization (Figure 3-11)
in which log intensity ratios M = log(Cy3/Cy5) = logCyG - logCy5 were
plotted against mean log intensities A = (logcya + logCy5)/2 for each
array spot as described by Yang et al., (2002). The resulting normalized
log intensities were then analyzed using the Mixed Model procedure of
SAS. The model included fixed effects of dye, treatment, adipose tissue
depot, and treatment x tissue interaction as independent variables. The
level of probability at which main effects and interactions were considered

significant was P < 0.05 and P < 0.10, respectively.

77

Results and Discussion

Northern blot analysis. Northern blot analysis was performed to
examine glucocorticoid receptor (a-iSOfOflTI) mRNA abundance between
the two adipose tissue depots and in response to dexamethasone
administration. Samples of RNA were hybridized with a 32P-Iabeled probe
specific for the 9-a subunit of the bovine glucocorticoid receptor.
Subcutaneous adipose exhibited higher abundance of GRa mRNA
message (P < 0.01) (Figures 3-1, 3-2) for GR than did intramuscular
adipose, even after adjustment for loading inefficiency through B-actin
normalization (Table 3-1). Additionally, it was observed that in both
adipose tissue depots, dexamethasone acutely down-regulated
expression of GRa mRNA (P < 0.05). These results are in agreement
with Preisler et al. (2000) who showed that bovine GRa proteins were
down-regulated in white blood cells in response to high glucocorticoid
concentrations at parturition. The northern blot analysis confirmed the
expression of GR in bovine adipose tissue as well as a response in gene
expression to dexamethasone treatment. Northern blot analysis for
expression of B-actin revealed additional bands within the intramuscualr
adipose samples that appeared to result from expression of a-actin. This
suggests that intramuscular adipose samples were most likely

contaminated with a small amount of muscle tissue from dissection.

78

cDNA microarray analysis. A number of genes were significantly
differentially expressed due to treatment with dexamethasone or location
of adipose tissue. The cDNA microarray analysis revealed genes that
changed expression in adipose tissue due to treatment with
dexamethasone. The most biologically significant gene that showed
differential expression upon stimulation by the glucocorticoid was the
urokinase receptor (P<0.01) (Figure 3-3). Expression of the urokinase
receptor has been associated with angiogenic function in epithelial cells
(Mignatti et al., 1991) and its expression appears to be induced by
increases in fibroblast growth factor (Mignatti et al., 1991; Pepper et al.,
1993). However, more recent work indicates that the urokinase receptor
and its associated cellular proteins also play a role in cell cycle control and
proliferation, and that expression of the urokinase receptor increases as
the cell cycle progresses (Zavizion et al., 1998). Glucocorticoid
stimulation increased urokinase receptor expression in both adipose
depots suggesting an increase in cell cycle activity of preadipose cells.
Assuming that the glucocorticoid was able to withdraw preadipocytes from
the proliferative cycle for differentiation, this increase in cell cycle activity
may be the result of the mitotic clonal expansion that precedes terminal
differentiation (Gregoire et al., 1998).

In addition to the urokinase receptor, several genes that are
involved in cell adhesion were differentially expressed in response to

glucocorticoid treatment. The glycoproteins P-selectin, CDG, CD28, and

79

GLYCAM-1 expression were all affected by dexamethasone
administration. The findings of Burton et al. (1995) and Burton and Kerhli
(1995) for similar cell adhesion molecule expression leukocytes showed a
pronounced down regulation by dexamethasone. In the current study,
C06 was down-regulated with DEX (P < 0.05), however expression of
CD28 and P-selectin were increased (both P < 0.05). The increase in
expression of P-selectin may be partially explained by the work of Xia et
al. (1998) who found P-selectin expression in cells is decreased by most
anti-inflammatory drugs, with the exception of glucocorticoids. This does
not, however, explain the apparent up-regulation of P-selectin expression.
It was also noted that GLYCAM-1, a gene that codes for a mucin-like
glycoprotein was differentially expressed between adipose tissue depot
and appeared to be differentially regulated by dexamethasone
administration. However biological function of this gene and its gene
product does not appear to be determined. An explanation of the
differential expression of genes that code for cell adhesion molecules in
adipose tissue is not apparent. However, because the products of these
genes are glycoproteins that are involved in cell membrane composition, it
is possible that the differential expression may be the result of changes in
cell size or number that were evoked by the dexamethasone treatment.
Differential gene expression was also observed between adipose
tissue depots for a number of genes. MDM2, a cellular protein that is

associated with the p53 tumor suppressor protein, had significantly greater

80

expression in intramuscular adipose (Figure 3—6). The p53 tumor
suppressor protein functions in the halting of cellular proliferation (Mulligan
and Jacks, 1998). The MDM2 protein acts as an oncogene by binding to
and inactivating p53 (Lundgren et al., 1997). Thus, the greater expression
of MDM2 in intramuscular adipose suggests that a larger percentage of
cells within that depot are still experiencing proliferation. Furthermore,
intramuscular adipose exhibited more abundant mRNA messages for
hypoxanthine phophoribosyltransferase (Figure 3-4), a rate-limiting
enzyme in purine synthesis. This suggests greater levels of nucleotide
synthesis and thus higher proliferative activity of cells in intramuscular
adipose. Additional evidence of a greater percentage of preadipose cells
in the intramuscular depot is shown by the greater expression (P < 0.05)
of alpha-tubulin in intramuscular versus subcutaneous adipose (Figure 3-
5). Alpha-tubulin is a protein involved in the cytoskeletal composition of
cells. The morphological changes in cell shape that accompany terminal
adipocyte differentiation are marked by a decrease in such structural
proteins as tubulin and actin (Spiegelman and Farmer, 1982).

Four different genes involved in cellular apoptosis were more highly
expressed in intramuscular adipose (P < 0.05). Caspase-4, a protein
involved in the fas-mediated pro-apoptotis pathway (Kamada et al., 1997;
Martin and Panja, 2002), pro-apoptosis regulator bax-alpha, and the
serine protease inhibitor p19 exhibited greater expression in intramuscular

tissue (Figures 3-10, 3—7, and 3-9, respectively). Additionally, mRNA for

81

TRAF 5, a factor associated with tumor necrosis factor-a (T NF-d) that
serves to mediate pro-death effects of this potent cytokine in signaling
pathways (Bradley and Pober, 2001), was more abundant in intramuscular

adipose (Figure 3-8).

82

Implications

The results of this study point to fundamental differences between
intramuscular and subcutaneous adipose on a molecular (gene
expression) level. However, differential gene expression in adipose tissue
due to depot location opens the possibility for independent manipulation of
adipose tissue depots.

Additionally, bovine adipose tissue expressed abundant
glucocorticoid receptor-alpha, especially in subcutaneous adipose tissue.
This suggests that glucocorticoids may provide a useful way to regulate

differential fat accretion in specific adipose depots.

83

Table 3-1. Densitometer quantifications of northern blots for GR9a and B-
actin

 

GR90 B—actin Adj. GR90

 

Sample Lane density density densitya
1-Subcutaneous, control 1 27.742 9.921 2.796
1-Subcutaneous, dex-treated 2 6.305 6.405 0.984
1-lntramuscular, control 3 7.261 8.565 0.848
1-lntramuscular, dex—treated 4 4.852 9.563 0.507
2-Subcutaneous, control 5 24.206 1 1 .342 2.134
2—Subcutaneous, dex-treated 6 14.873 8.766 1.697
2-lntramuscular, control 7 9.041 8.712 1.037
2-lntramuscular, dex-treated 8 5.1 50 8.946 0.576

 

alGR90L density divided by B-actin density

84

Figure 3-1. Northern blots: Glucocorticoid receptor 9-alpha subunit mRNA
(tap); beta-actin (bottom) for subcutaneous (SQ) or intramuscular (IM)
adipose tissue treated with saline (C) or dexamethasone (D).

 

 

SQ IM SQ IM
C D C

1.28kb

2.8kb

 

85

Figure 3—2. Northern blot densitomer quantifications for glucocorticoid
receptor-alpha mRNA abundance in intramuscular (IM) and subcutaneous
(SQ) adipose tissue from steers treated with saline (CON) or
dexamethasone (DEX)

 

 

 

 

GR 9-alpha mRNA abundance

 

 

 

SQ-CON SQ-DEX lM-CON lM-DEX

a,b,c-means without a common letter significantly differ (P < 0.05).

86

Figure 3-3. Expression of mRNA for urokinase receptor in intramuscular
(IM) and subcutaneous (SQ) adipose tissue from steers treated with saline
(CON) or dexamethasone (DEX)

Urokinase receptor

 

1.4 __._

 

 

 

1.27

0.8 ~

0.6-7

mRNA abundance

0.4777

0.2

 

 

 

0 2

IM- CON SQ-CON lM-DEX SQ-DEX

a,b— means without a common letter significantly differ (P < 0.05).

87

Figure 3-4. Expression of mRNA for hypoxanthine
phosphoribosyltransferase in intramuscular (IM) and subcutaneous (SQ)
adipose tissue from steers treated with saline (CON) or dexamethasone

(DEX)

Hypoxanthine phosphoribosyltransferase

 

1.2 ——

mRNA abundance

 

 

 

lM-CON SQ-CON IM-DEX SQ-DEX

a,b- means without a common letter significantly differ (P < 0.05).

88

Figure 3-5. Expression of mRNA for alpha tubulin in intramuscular (IM)
and subcutaneous (SQ) adipose tissue from steers treated with saline
(CON) or dexamethasone (DEX)

Alpha-tubulin

 

1.2 -—————— a a

mRNA abundance

 

 

 

 

lM-CON SQ-CON lM-DEX SQ-DEX

a,b- means without a common letter significantly differ (P < 0.05).

89

Figure 3-6. Expression of mRNA for MDM2 in intramuscular (IM) and
subcutaneous (SQ) adipose tissue from steers treated with saline (CON)
or dexamethasone (DEX)

MDM2

 

1.4

 

 

mRNA abundance

 

 

 

IM-CON SQ-CON lM—DEX SQ-DEX

a,b- means without a common letter significantly differ (P < 0.05).

90

Figure 3-7. Expression of mRNA for Apoptosis regulator bax—alpha in
intramuscular (IM) and subcutaneous (SQ) adipose tissue from steers
treated with saline (CON) or dexamethasone (DEX)

Apoptosis regulator bax-alpha

 

 

 

mRNA abundance

 

 

 

 

lM-CON SQ-CON IM-DEX SQ-DEX

a,b— means without a common letter significantly differ (P < 0.05).

91

Figure 3-8. Expression of mRNA for TRAF5 in intramuscular (IM) and
subcutaneous (SQ) adipose tissue from steers treated with saline (CON)

or dexamethasone (DEX)

TRAF 5

 

mRNA abundance

 

 

 

lM-CON SQ-CON lM-DEX SQ-DEX

a,b— means without a common letter significantly differ (P < 0.05).

92

 

Figure 3-9. Expression of mRNA for serine protease inhibitor p19 in
intramuscular (IM) and subcutaneous (SQ) adipose tissue from steers
treated with saline (CON) or dexamethasone (DEX)

Serine protease inhibitor p19

 

1.4 a

 

1.2 «r

0.8 -

 

0.6 7

mRNA abundance

0.4 -

0.2 «

 

 

 

 

IM-CON SQ-CON lM-DEX SQ-DEX

a,b- means without a common letter significantly differ (P < 0.05).

93

Figure 3-10. Expression of mRNA for Caspase-4 in intramuscular (IM) and
subcutaneous (SQ) adipose tissue from steers treated with saline (CON)
or dexamethasone (DEX)

Caspase-4

 

1.4

 

1.2 —

 

0.8 -

0.6 ~

mRNA abundance

0.4 .

0.2 -

 

 

 

 

0 a

lM-CON SQ-CON lM-DEX SQ-DEX

a,b- means without a common letter significantly differ (P < 0.05).

94

Figure 3-11. M-A scatterplot of data from array comparing intramuscular,
control tissue vs. intramuscular, dexamethasone-treated tissue before and
after normalization

-0.
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3 . 20000 3 .40000 3 . 80000 3 . 80000 4 . 00000 4 . 20000 4 .40000 4 . 80000 4 . 80000

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95

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98

CHAPTER IV

INTERPRETIVE SUMMARY

Adipose tissue of beef cattle, its rate of development, and its cost of
accretion, form a oomerstone of profitability in the current beef industry.
Accumulation of subcutaneous fat has historically been used as an
indicator of quality in live cattle and as a tool to determine a market end
point for fed slaughter cattle. However, actual correlation of quality to
subcutaneous fat levels has been shown to be inconsistent at best, to
highly ineffective at worst. In addition, the development of subcutaneous
fat requires a greater input of dietary energy than lean growth and thus
comes at the expense of decreased feed efficiency. From a carcass
standpoint, increases in subcutaneous adipose require greater trim and
result in a lower percentage of carcass weight that goes into retail product,
decreasing revenues. In contrast, increased intramuscular adipose in
beef carcasses is desirable, and it is the amount of visible intramuscular
fat or marbling that is the primary determinant of quality grade in beef.
Because cattle in the US. do not have sufficient marbling to meet
consumer demand in the current industry, decreased revenues are the
result. For these reasons, the focus of research and production has
centered around the goal of decreasing subcutaneous fat while
concurrently increasing intramuscular fat. The realization of this challenge

has been made more difficult by the lack of response of intramuscular fat

99

to manipulation through management, nutrition, or physiology. Despite
the difficulty of influencing intramuscular fat deposition, studies have
shown that intramuscular and subcutaneous adipose tissue exhibit unique
characteristics when compared on a cellular level. Therefore, the
objectives of the current studies were to evoke and characterize
differential responses between two adipose tissue depots. The purpose of
the objectives was one of discovery and meant to provide a point of origin
for further investigations on differential adipose tissue accretion.

In the initial experiment fat deposition of early weaned beef steers
was examined in response to administration of dexamethasone therapy
and supplementation of sunflower oil in the diet. Previous work had
pointed to a differential response of intramuscular and subcutaneous
adipose to glucocorticoid exposure. Past research also indicated the
effects of glucocorticoids may be mediated through an increase in
arachadonic acid metabolites, namely prostacyclins. Arachadonic acid is
synthesized from the precursor linoleic acid, which is found in high
concentrations in sunflower oil. Thus the treatments were chosen to form
a 2 x 2 factorial study.

Ultrasound readings were taken on steers throughout the study to
monitor fat deposition during the course of the trial. The results of the
ultrasound showed that dexamethasone did not elicit a significant
response in intramuscular tissue. Dexamethasone did lower

subcutaneous fat over the trial but not significantly. Over the duration of

100

the experiment, the supplementation of sunflower oil did show an increase
in intramuscular fat but again, it was not to a significant degree. Despite
the positive results of the ultrasound, significant differences did not exist in
final carcass characteristics for quality or yield. Additionally, it was
determined that the fatty acid profile of steaks from the steer carcasses
was not significantly affected by treatment.

Although the treatments appeared to stimulate a response in
adipose tissue, the results of the end measurements of the study were
inconclusive. This may have been confounded by an extended finishing
period for the steers (138 d) after the cessation of treatment. Overall, the
results of the study point to dexamethasone and sunflower oil as possible
tools for the manipulation of adipose tissue in fed beef cattle. However,
the time, duration, and dosage of dexamethasone for optimum response
require further investigation for determination.

In the second experiment, intramuscular and subcutaneous
adipose tissue samples were obtained from four fed beef steers at
slaughter. Steers were administered a single injection of either saline or
dexamethasone 24 h before harvest. Messenger RNA was extracted from
tissue samples and differential gene expression between treatment groups
and between adipose depot location was determined by cDNA microarray
analysis and Northern blot hybridization. One gene that was differentially
expressed upon stimulation of the glucocorticoid was the urokinase

receptor. The urokinase receptor and its associated cellular proteins play

101

a role in cell cycle control and proliferation and expression of the
urokinase receptor increases as the cell cycle progresses. Glucocorticoid
stimulation increased urokinase receptor expression in both adipose
depots suggesting an increase in cell cycle activity of preadipose cells. If
the glucocorticoid was able to withdraw preadipocytes from the
proliferative cycle for differentiation, this increase in cell cycle activity may
be the result of the mitotic clonal expansion that precedes terminal
differentiation.

Differential gene expression was also observed between adipose
tissue depots for a number of genes. MDM2, a cellular protein that acts
as an oncogene by binding to and inactivating p53 tumor suppressor gene
exhibited greater expression in intramuscular adipose suggesting a larger
percentage of cells within that depot are still experiencing proliferation.
Furthermore, intramuscular adipose exhibited more abundant mRNA
messages for hypoxanthine phophoribosyltransferase, a rate-limiting
enzyme in purine synthesis, further indicating proliferative activity of cells.
Additional evidence of a higher percentage of preadipose cells in the
intramuscular depot is shown by the differential expression of alpha-
tubulin. The significantly higher expression of structural proteins such as
alpha-tubulin in intramuscular tissue, suggests a less mature, preadipose
cell type.

In addition there were four genes that were differentially expressed

between depots that are associated with cellular apoptosis. These

102

 

included pro-apoptosis regulator bax-alpha, serine protease inhibitor p19,
Caspase-4, and TRAF5. Each of these genes had a significantly greater
expression in intramuscular adipose tissue suggesting the cells within that
depot are undergoing a greater rate of apoptosis.

Northern hybridization of mRNA with a radioactive probe specific
for the 9-cr subunit of the glucocorticoid receptor (GR), confirmed its
expression in both adipose locations. Additionally, it was shown that GR
expression was greater in subcutaneous adipose, and that
dexamethasone treatment reduced expression of GR in both types of
adipose. The results show that not only was dexamethasone successful
in evoking a response in adipose tissue from both depots, but also that the
differential level of GR expression may provide a means for separate
manipulation of intramuscular and subcutaneous adipose depots.

The results of the study point to glucocorticoids as a potential
endocrine regulator in adipose that has the capability to elicit a response
that is unique to adipose depot location. Furthermore the identification of
the urokinase receptor gene as a possible target of glucocorticoid-induced
response provides a clue in the elucidation of the pathways involved in
dexamethasone-mediated differentiation. Identification of the differential
expression of MDM2 between intramuscular and subcutaneous adipose,
gives a potential subject for further study of biological differences between
the two tissues. Additionally, with the differential gene expression taken

as a whole, itprovides a new perspective on the two different adipose

103

tissue depots as unique cell populations. With intramuscular adipose
tissue representing a group of cells that have greater rates of proliferation
and apoptosis, these cells appear to have a greater turnover of cells. In
contrast, cells from subcutaneous tissue appear to represent a more
stable cell population in which lipid metabolism is a more primary function
of the cells. Further investigations into the exact function of specific genes
in the molecular pathways of adipocyte growth and metabolism will be
required before any practical uses of their differential expression can be
realized in adipose tissue development. However, it does provide a new
aspect for research in this area of production as well as a greater
understanding of the biological function of the different adipose tissue

depots.

104