Download ENERGY INTAKE DURING THE DRY PERIOD AFFECTS

ENERGY INTAKE DURING THE DRY PERIOD AFFECTS
EXPRESSION OF INFLAMMATION-ASSOCIATED GENES IN
THE LIVER DURING THE TRANSITION PERIOD
Nicole A. Janovick Guretzky, Juan J. Loor, Heather M. Dann,
and James K. Drackley
TAKE HOME MESSAGES
•
Non-specific inflammatory responses around the time of calving have been observed in
cows with fatty liver.
•
An inflammatory response was observed around the time of calving for cows in this
study, regardless of their dietary energy intake during the dry period.
•
Evidence for alteration in the onset and duration of the inflammatory response was
observed for cows whose energy intake was restricted in the dry period compared to cows
fed excess energy or cows fed to their energy requirement.
INTRODUCTION
During the transition period, cows mobilize adipose stores to supply energy to organs and for
production of milk. Mobilization of adipose results in a large flux of non-esterified fatty acids
(NEFA) in the blood that are processed by the liver. If the liver cannot process these fatty acids
quickly enough, then fat accumulates in the liver tissue. Enzymes that influence the rate of
clearance of fat from the liver and slow down the mobilization of fat from body adipose stores
are made from genes under the control of transcription factors known as peroxisome proliferator
activated receptors (PPAR). PPAR-α is found in liver tissue, and when activated, increases the
activity of enzymes that increase the rate of fatty acid oxidation for NEFA coming to the liver.
PPAR-γ is found in large amounts in adipose tissue and when activated, slows down the rate of
adipose mobilization by signaling the adipose cells to store more energy rather than mobilize it.
Taken together, the activation of these PPAR’s would theoretically result in more fat cleared
from liver and less fat mobilized from adipose tissue.
When injury occurs in the body, a general, non-specific inflammation occurs to prepare for
fighting infection and to speed the healing process. This inflammatory response results in the
production of key ‘messenger proteins’ by immune cells and other tissues. In turn, these
messenger proteins signal the liver and immune cells to start producing proteins to activate the
immune system. Two of these important first messengers, or cytokines, are tumor necrosis
factor-α (TNF-α) and interleukin-6 (IL6). An inflammatory response has been reported in cows
around the time of calving and into early lactation. Although this could be a normal response to
the large cortisol surge that occurs at calving, others have noted that the degree of this
inflammation has been associated with the development of fatty liver syndrome in cows.
Interestingly, inflammatory cytokines such as TNF-α and IL6 inhibit the expression of PPAR’s.
Therefore, more inflammatory cytokine production could result in less clearance of fat from liver
and increased mobilization of adipose, which together would contribute to development of fatty
liver.
Although a relationship exists between depressed immune function and the transition period, it is
not known if and how the inflammatory response could be manipulated through dietary
strategies. The objective of this study was to measure changes in the inflammatory-associated
genes described above in liver tissue from cows fed three levels of energy from the dry period
through calving. The objective was to determine if there was evidence for a dietary effect on
occurrence or duration of the inflammatory response at calving.
METHODS
Messenger ribonucleic acid, or mRNA, produces the ‘code’ in cells to direct the synthesis of
proteins. Each protein is coded by a specific gene. Therefore, if there is an increase of
abundance of mRNA in cells, then the abundance of the protein coded for by that gene is also
presumed to increase. We studied the abundance of mRNA as an indirect measure of the
inflammatory-associated proteins in liver.
A subset of cows from a larger project (Dann, 2004 PhD Thesis) was chosen for this experiment.
Thirteen Holstein cows entering their second or greater lactation consumed one of three far-off
diets from dry-off through three weeks prior to expected calving. The control diet was
formulated to meet 100% of National Research Council (NRC, 2001) recommendations for
energy (Control). A higher nutrient density diet was fed either for ad libitum intake, which
provided 180% of calculated energy requirement (Excess) or at restricted intake to provide 80%
of the energy requirement (Restricted). Three weeks prior to expected calving, cows were
offered a close-up ration at either restricted (80% of requirement) or ad libitum intake until
calving. After calving, all cows were fed a common lactation ration, balanced according to NRC
recommendations (NRC, 2001). Cows had adequate body condition before calving (BCS = 2.9 +
0.50).
Total RNA was isolated from liver tissue obtained by biopsy on day -65, -30, -14, +1, +14, +28,
and +49 relative to calving. The relative abundance of mRNA in each of the samples was
measured by ‘real time’ polymerase chain reaction (PCR). This technology makes many copies
of certain regions in the mRNA that correspond to the genes of interest. By using a ‘control’ or
‘reference’ gene that is normally present in liver and does not change with production stage or
disease state, we were able to calculate the difference between the reference gene and the genes
we were interested in. These changes were then compared to the value obtained at day -65
relative to calving. A fold change was calculated to get the increase or decrease in abundance of
the mRNA coding for a particular gene relative to day -65.
RESULTS AND DISCUSSION
A peak was observed in TNF-α one day postcalving for the cows in the Control and Restricted
groups, and at 14 days postcalving for cows in the Excess group. This peak was a four-fold
increase (P < .001) over the observed abundance in TNF-α observed at dry-off in the Control
cows. Additionally, this peak was greater than in either the Excess or Restricted groups at day
one postcalving (P < .001). Although no clear peaks were observed for IL6 in any of the groups,
the relative abundance of RNA was elevated in the weeks surrounding calving. Taken together,
both of the changes observed would be indicative of a general inflammatory response around the
time of calving that appeared to be influenced somewhat by the diet fed in the dry period.
A peak for PPAR-α occurred one day postcalving for cows in the Control and Restricted groups,
but there was little change in relative abundance of PPAR-α in the Excess group. This
corresponded to the time when NEFA were highest in the blood and when the need for oxidation
of these fatty acids in the liver would have been greatest. If Restricted cows already had
increased oxidation occurring in the liver as a result of dietary energy restriction, perhaps this
could explain why a change relative to dryoff was not observed. PPAR-γ peaked on day one
postcalving for the Restricted group; however, peaks for the Control and Excess group occurred
on day 14 after calving. Because the peaks for PPAR-γ were not as pronounced and occurred
after TNF-α abundance had decreased, this could indicate an influence of this cytokine on the
expression of PPAR-γ (and PPAR-α). Taken together, these changes are consistent with a
greater likelihood for fat accumulation in the liver around calving for cows that were overfed
energy during the dry period.
IL6
4.5
4.0
Fold change relative to d -65
Fold change relative to d -65
TNF-alpha
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0.0
-65
-30
-14
1
14
Day relative to calving
28
49
4.5
4.0
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0.0
-65
-30
-14
1
14
Day relative to calving
28
49
PPAR gam m a
4.5
Control
4.0
Excess
3.5
Restricted
3.0
2.5
2.0
1.5
1.0
0.5
0.0
-65
-30
-14
1
14
Day relative to calving
28
49
4.5
Fold change relative to d -65
Fold change relative to d -65
PPAR alpha
4.0
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0.0
-65
-30
-14
1
14
28
49
Day relative to calving
Figure 1. Changes in mRNA abundance relative to dry-off (day -65) for tumor necrosis factor-alpha
(TNF-α), interleukin-6 (IL6), and peroxisome proliferator activated receptor-γ (PPAR γ) and –α (PPAR
α). Cows were fed 100% (Control), 180% (Excess), or 80% (Restricted) of their energy requirement
(NRC, 2001) through the dry period and a common lactation ration after calving. The four-fold increase
in abundance for TNF-α one day post-calving in the Control group was significant when compared to d 65 (P < .001). The mRNA abundance one day post-calving for the Control group was also greater than
for the Excess or Restricted groups (P < .001) at day one post-calving. Other comparisons were not
statistically significant.