Download MILK SYNTHESIS ENZYMES AND THEIR ROLES IN MILK QUALITY

MILK SYNTHESIS ENZYMES AND
THEIR ROLES IN MILK QUALITY
Massimo Bionaz and Juan J. Loor
TAKE HOME MESSAGES
•
The presence of trans-fatty acids (tFA) in bovine milk has increased awareness in the
health-conscious consumer. Cow’s milk, despite its high tFA content, is still considered
a wholesome food because of the high conjugated linoleic acid (CLA) as well as presence
of omega-3 FA, which have a demonstrated anticarcinogenic activity.
•
Increased CLA concentration in milk during lactation is accompanied by an overall
decrease in fat yield (“milk fat depression”). Thus, increasing CLA without decreasing
milk fat yield requires deep understanding of the mechanisms governing synthesis of
milk fat.
•
Our research demonstrates that mammary gland contains large amounts of the enzyme
needed for CLA synthesis, and its abundance is increased during lactation suggesting a
crucial role for this enzyme in milk fat synthesis as well.
•
Synthesis of CLA is not only driven by the amount of enzyme but by the coordination
among many proteins involved in milk fat synthesis, and also the presence of substrate
for these enzymes. The latter could be manipulated through dietary management.
INTRODUCTION
CLA intake in humans is of interest because of the potential health benefits this fatty acid (FA)
may confer. The anticarcinogenic activity of CLA has been clearly established for a wide range
of cancer types. Additional health effects were discovered for CLA. The predominant source of
CLA in human diets is ruminant-derived food products, with dairy products contributing about
75% of the total; hence, research has concentrated on increasing the CLA content per unit of fat
in milk. CLA can originate as intermediates during rumen biohydrogenation of linoleic and
linolenic acid (highly abundant in fresh forage). The pathway of rumen biohydrogenation of
unsaturated fatty acids goes from linoleic (cis9,cis12 18:2) → CLA (cis9,trans11 18:2) → transFA (trans11 18:1) → stearic acid (18:0). Most of those intermediates escape the rumen and are
taken up by the mammary gland and inserted into milk fat. Trans11 18:1 and 18:0 can be
transformed in the mammary gland into cis9,trans11 18:2 (CLA) by the enzyme ∆9 desaturase.
The gene symbol for this enzyme is SCD for stearoyl-CoA desaturase. Increasing activity of this
enzyme is desirable in order to obtain larger amounts of CLA in milk fat.
Milk fat contains omega-3 FA, which also have proven to elicit positive effects on human health.
Dietary omega-6 fatty acids also are important because most of those are considered “essential”
FA (i.e. not synthesized by the organism, but with essential functions). Western diets typically
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have an omega-6 to omega-3 ratio of 20–30:1, whereas the ideal ratio is thought to be 4:1 or less.
Thus, an important goal for dairy producers is to enrich milk with omega-3 FA. The mammary
gland contains two enzymes called fatty acid desaturase 1 and 2 (or FADS1 and FADS2). FA
products of these enzymes are referred to as omega-6.
As part of our research program in the regulation of milk fat synthesis, we measured the pattern
of gene expression of desaturases in mammary gland during lactation, and their relationship with
the composition of fatty acids in milk fat. A primary goal is to define the role of those enzymes
in relationship with other enzymes associated with milk fat synthesis. In the long-term, results
from this research can help in manipulating diet to obtain maximum effects on the yield of
“beneficial” milk FA (CLA, omega-3, omega-6). Results also appear to challenge the current
concept of gene-assisted selection based on one of few genes. Instead, selection of cows for
high-quality milk fat should be based on potential expression of several key enzymes.
METHODS
Six high producing Holstein dairy cows from the University of Illinois dairy facility were
followed during the entire lactation. The cows received a typical diet for lactating cows
composed (% dry matter) of 28% corn silage, 20% Alfalfa silage, 10% cottonseed and 42%
concentrate. Mammary tissue was biopsied at 15 days before calving, and at 1, 15, 30, 60, 120,
and 240 days after calving. Tissue gene abundance (“mRNA”) of several enzymes involved in
milk fat synthesis plus SCD, FADS1, and FADS2 was measured. Milk yield was measured
during the entire lactation. Milk samples were collected the day before biopsy and fat and fatty
acid composition analyzed.
RESULTS AND DISCUSSION
50.00
35.0
40.00
% mRNA abundance
40.0
30.0
2.0
1.5
Fat yield
Milk Yield
20.00
10.00
SCD
FASN
ACACA
240
120
Day relative to parturition
FADS2
0.05
0.00
0.5
60
30.00
FADS1
1.0
15
30
kg/d
The curve of lactation was typical of high producing dairy cows and the milk fat yield reached
peak at 30 d (Fig. 1). SCD was the most abundant among all genes measured (Fig. 1), FADS2
was almost undetectable and FADS1 had low expression in mammary tissue. All genes had a
similar pattern with peak in expression at 60 d (Fig. 2). Genes involved in “de novo synthesis”
and SCD had a large up-regulation during lactation. ∆5 desaturase (FADS1) also had a large
increase (18-fold at 60 d), while ∆6 desaturase (FADS2) increased <3-fold.
Figure 1 – Milk and fat yield (kg/d) during the entire lactation, and percentage mRNA abundance in
mammary tissue of genes involved in de novo synthesis (ACACA or acetyl-CoA carboxylase and FASN or
fatty acid synthase) and desaturation of fatty acids.
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0.11
0.6
0.014
0.5
0.012
0.4
0.010
0.09
0.45
0.08
0.40
0.07
0.35
0.006
15
30
240
120
60
0.008
Δ5 activity
Δ6 activity
0.2
0.06
15
30
0.30
0.3
240
0.60
120
0.10
0.62
60
0.64
0.016
Δ5 activity
18:0 & t18:1 Δ9 activity
0.66
0.7
14:0 & 16:0 Δ9 activity
Δ9 18:0
Δ9 t18:1
Δ9 14:0
Δ9 16:0
0.68
0.12
Δ6 activity
Δ5 and Δ6 desaturase indexes
Δ9 desaturase indexes
0.70
Day relative to parturition
Day relative to parturition
Figure 3. The Δ9 desaturase indexes include the Δ9 activity on 18:0, on trans11 18:1, on 14:0, and on 16:0.
The Δ5 and Δ6 indexes also are reported.
The ratio among yield of omega-3/omega-6 FA had a linear decrease during lactation (Fig.2).
The overall ∆9 desaturase activity (Table 1) decreased during lactation with a minimum at 120 d.
However, the specific activity of ∆9 desaturase on all FA except palmitic acid tended to increase
during lactation (Fig. 3)., with the larger increase on activity toward trans11 18:1 to generate
cis9,trans11 18:2 (or CLA). ∆5 desaturase activity had a peak at 30 d and decreased thereafter.
∆6 activity had a large increase during lactation. Indexes of desaturase activity did not have a
direct relationship with the pattern in gene expression of the enzymes. However, the relative
amount of mRNA agreed with the amount of product of each enzyme. In fact, the amount of ∆9
desaturase (or SCD) products are 20% of total milk fatty acids, while the FADS1 and FADS2
products accounts for 0.12% and 0.02%.
Palmitic acid (16:0) and oleic acid (cis9 18:1) are the most abundant fatty acid in milk.
Furthermore, milk from these cows also had good amounts of CLA (cis9,trans11 18:2), and
omega-3 (18:3n3 + 20:5n3). Daily milk yield of CLA and 20:5n3 increased during lactation
while 18:3n3 decreased. Overall, data suggest that the activity of the ∆9 desaturase enzyme is not
only a consequence of gene expression, but the presence of the substrate and the activity of other
proteins is important as well.
Ratio n3/n6
1.4
1.2
1.0
0.8
Day relative to parturition
240
120
60
15
30
240
120
60
0.6
-15
1
15
30
240
120
60
Day relative to parturition
1.6
SCD
FADS1
FADS2
48
ACACA
42
FASN
36
30
24
18
12
6
3
2
1
-15
1
15
30
Fold change relative to -15 d
9
8
7
6
5
4
3
2
1
0
Omega-3/Omega-6
FA desaturation
FA synthesis
Day relative to parturition
Figure 2 – Expression patterns of key genes involved in fatty acid synthesis (ACACA, FASN), and desaturation
(SCD, FADS1, FADS2). Left is reported the ratio between Omega-3/Omega-6 FA
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Table 1. Yield (mmole/d) of selected fatty acids throughout the entire lactation. Letters abc
denote differences between day of lactation.
Fatty Acid
16:0
16:1cis9
18:0
18:1cis9
18:1trans11
18:2cis9,cis12
18:2cis9,trans11
18:3n3
18:3n6
20:3n6
20:4n6
20:5n3
Δ9-Desaturase index
15
1515
158.3*
755.2bc
1207bc
23.4
164.3
11.9a
22.0bc
1.6
7.3
7.9
0.5a
0.35b
Day Relative to Calving
30
60
120
1834
1669
1488
156.2*
120.8
103.7*
854.3c
659.4
432.7a
1410c
1059
743a
26.0
25.2
20.9
216.1b
168.3
131.1a
16.9
16.8
17.2
26.5c
20.0
16.1ab
1.9
2.0
1.9
6.8
7.5
7.6
9.4b
7.6
6.5a
1.2b
1.3b
1.0
0.33b
0.30
0.27a
240
1403
104.2
486.6ab
848ab
25.4
136.7a
20.5b
12.7a
2.2
10.0
8.1
0.7
0.30
P-Value
0.23
0.03
<0.01
<0.01
0.68
0.02
0.07
<0.01
0.46
0.26
0.08
<0.01
<0.01
SUMMARY AND CONCLUSIONS
Our research group is the first to measure gene expression of desaturase enzymes during the
entire lactation in dairy cows. ∆9 desaturase (SCD) is extremely high in mammary tissue and has
a large increase in mRNA abundance at peak lactation, suggesting a crucial role of this enzyme
both in milk fat synthesis and CLA synthesis. The other two desaturases appear to play minor
roles compared with SCD. Milk fatty acid data for oleic acid and CLA vs. omega-3 support this
conclusion. The similar pattern of expression for key genes involved in milk fat synthesis and
desaturation suggests a necessity for coordinated regulation of all those proteins to accomplish
the task of synthesizing milk fat. The lack of relationship in the pattern of desaturases gene
expression and indexes of desaturase activity suggest that the latter cannot be used to estimate
the first, as it is widely believed. Another factor playing a role in the output of “beneficial” FA
is the amount of substrate, and this can be easily manipulated though diet. Our data suggest that
the supplementation of precursors for CLA and omega-3 (e.g., oil seeds, oils) can not be done
randomly. That is, to maximize the efficiency of CLA synthesis dietary FA precursors should be
provided when the desaturases and other enzymes involved in milk fat synthesis are at or close to
peak expression (i.e. at 60 d post-calving). In addition, we propose that selection of dairy cows
based on a single gene (e.g. SCD) will probably not improve milk quality (e.g. CLA). In this
context, data suggest that in the future selection based on several genes involved in milk fat
synthesis might be more effective.
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