Endogenous production of omega-3 fatty acids in milk · •Omega-3 (ω3) polyunsaturated fatty...
Transcript of Endogenous production of omega-3 fatty acids in milk · •Omega-3 (ω3) polyunsaturated fatty...
Animal Biotechnology and Genomics Education
Endogenous production of omega-3 fatty acids in milk
Alison Van Eenennaam, Ph.D.
Cooperative Extension Specialist Animal Biotechnology and Genomics
Phone: 530 752-7942
Animal Biotechnology and Genomics Education
Overview
• The importance of omega-3 (aka ω3 or n3) polyunsaturated fatty acids (n3 PUFAs)
• Pathways of biosynthesis • The problem with ruminants • A biotech work around • In vitro results • In vivo results • Road blocks • Conclusions
Animal Biotechnology and Genomics Education
What is an omega-3 fatty acid ?
In the omega system the numbering refers to the first carbon with a double bond relative to the carbon at the methyl end of the molecule.
Animal Biotechnology and Genomics Education
Pathways of polyunsaturated fatty acid synthesis. Enzymes are indicated in ovals and major
products are boxed
18:2n6 20:3n6 18:3n3 20:4n3
18:3n6 20:4n6 18:4n3 20:5n3
Δ5/Δ6
fat-2
Δ5/Δ6
fat-1
Zebrafish
Modification of milk fatty acid composition
Vertebrates lack
the fatty acid desaturase
enzymes required for
the synthesis of linoleic
acid (LA, 18:2n-6) and
alpha-linolenic acid
(ALA, 18:3n-3), and are
therefore dependent on
dietary sources to obtain
these essential PUFA.
The nematode
Caenorhabditis elegans
is able to synthesize ALA
from LA by virtue of an
endogenous omega-3
fatty acid desaturase
Animal Biotechnology and Genomics Education
The diet of our ancestors was higher in fiber, rich in fruits, vegetables, lean meat, and fish.
The diet was less dense in calories, lower in total fat and saturated fat, and contained approximately equal amounts of ω6 and ω3 PUFAs
In Western diets, the current ratio of ω6 to ω3 PUFAs is about 10 to 20:1, indicating that Western diets are deficient in ω3 fatty acids compared with
the diet on which humans evolved
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ω3 is good for me
• Omega-3 (ω3) polyunsaturated fatty acids (PUFA) are well documented for their beneficial health effects
• Used to treat and prevent coronary heart disease, cancer, metabolic syndrome, Crohn disease, rheumatoid arthritis, inflammatory and immune-based diseases, autoimmune diseases, even schizophrenia
• The average intake of ω3 PUFA is about 1.6 grams/day in the United States. Nutrition scientists suggest the daily intake should be closer to 2.85 grams/day
ω3
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Which would you rather eat to
lower your risk of heart disease ?
A. B.
Omega-3 dairy products coming soon.....
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Modification of milk fat to benefit human health
• Ruminant milk is high in saturated fat and does not contain high levels of PUFAs as a result of rumen biohydrogenation
• The incorporation of low levels (400 mg/day) of ω3 PUFAs in milk has been found to have significant human health benefits
• How can the fatty acid composition of milk be improved to benefit human health ?
ω3
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Fatty acid composition of
bovine milk
Environmental approaches
• Bypass rumen
• Add it to the milk
Genetic approach
• Provide desaturases
(ω3)
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Hypothetical effect of desaturases on
fatty acid composition of bovine milk
18:1n-9
18:3n-3
ALA
18:2n-6 LA
FAT-2
FAT-1
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Phospholipid vs. Triacylglycerol
The C. elegans Fat 1 omega-3 desaturase operates on acyl groups that are esterified to phospholipids and so it is called an acyl-lipid desatuarase (rather than a CoA desaturase)
Animal Biotechnology and Genomics Education
In vitro proof of concept – Kerri Morimoto (MS)
CMV promoter fat-2fat-1 TK polyAEF-1 alpha promoterSV40 polyACMV promoter fat-2fat-1 TK polyAEF-1 alpha promoterSV40 polyA
• Two desaturases cloned into pBUD • Adenovirus transfected into HC11 • Analyzed fatty acid composition of transfected cells three days after transfection
Omega-3 Delta-12
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0
2
4
6
8
10
12
14
16
18
LacZ Control
Fat-1/Fat-2
b
0
2
4
6
8
10
C16:1 C18:1n-9 C18:2 n-6 (LA) C18:3 n-3 (ALA) C20:4 n-6 (AA) C20:5 n-3 (EPA)
Fatty Acids
b
Mol %
Fatty A
cid
s
18:1n-9
18:3n-3
ALA
18:4n-3 20:4n-3 20:5n-3
EPA
20:4n-6
AA
20:3n-618:3n-618:2n-6
LA
FAT-2
FAT-1 FAT-1 FAT-1 FAT-1
18:1n-9
18:3n-3
ALA
18:4n-3 20:4n-3 20:5n-3
EPA
20:4n-6
AA
20:3n-618:3n-618:2n-6
LA
FAT-2
FAT-1 FAT-1 FAT-1 FAT-1
a
0
2
4
6
8
10
12
14
16
18
LacZ Control
Fat-1/Fat-2
b
0
2
4
6
8
10
C16:1 C18:1n-9 C18:2 n-6 (LA) C18:3 n-3 (ALA) C20:4 n-6 (AA) C20:5 n-3 (EPA)
Fatty Acids
b
Mol %
Fatty A
cid
s
18:1n-9
18:3n-3
ALA
18:4n-3 20:4n-3 20:5n-3
EPA
20:4n-6
AA
20:3n-618:3n-618:2n-6
LA
FAT-2
FAT-1 FAT-1 FAT-1 FAT-1
18:1n-9
18:3n-3
ALA
18:4n-3 20:4n-3 20:5n-3
EPA
20:4n-6
AA
20:3n-618:3n-618:2n-6
LA
FAT-2
FAT-1 FAT-1 FAT-1 FAT-1
a Phospholipids
Triaclyglycerides
Pathways of polyunsaturated fatty acid synthesis. Enzymes are indicated in ovals and major
products are boxed
18:2n6 20:3n6 18:3n3 20:4n3
18:3n6 20:4n6 18:4n3 20:5n3
Δ5/Δ6
fat-2
Δ5/Δ6
fat-1
Zebrafish
0
2
4
6
8
10
12
14
16
18
LacZ Control
Fat-1/Fat-2
b
0
2
4
6
8
10
C16:1 C18:1n-9 C18:2 n-6 (LA) C18:3 n-3 (ALA) C20:4 n-6 (AA) C20:5 n-3 (EPA)
Fatty Acids
b
Mo
l %
Fa
tty A
cid
s
18:1n-9
18:3n-3
ALA
18:4n-3 20:4n-3 20:5n-3
EPA
20:4n-6
AA
20:3n-618:3n-618:2n-6
LA
FAT-2
FAT-1 FAT-1 FAT-1 FAT-1
18:1n-9
18:3n-3
ALA
18:4n-3 20:4n-3 20:5n-3
EPA
20:4n-6
AA
20:3n-618:3n-618:2n-6
LA
FAT-2
FAT-1 FAT-1 FAT-1 FAT-1
a
0
2
4
6
8
10
12
14
16
18
LacZ Control
Fat-1/Fat-2
b
0
2
4
6
8
10
C16:1 C18:1n-9 C18:2 n-6 (LA) C18:3 n-3 (ALA) C20:4 n-6 (AA) C20:5 n-3 (EPA)
Fatty Acids
b
Mo
l %
Fa
tty A
cid
s
18:1n-9
18:3n-3
ALA
18:4n-3 20:4n-3 20:5n-3
EPA
20:4n-6
AA
20:3n-618:3n-618:2n-6
LA
FAT-2
FAT-1 FAT-1 FAT-1 FAT-1
18:1n-9
18:3n-3
ALA
18:4n-3 20:4n-3 20:5n-3
EPA
20:4n-6
AA
20:3n-618:3n-618:2n-6
LA
FAT-2
FAT-1 FAT-1 FAT-1 FAT-1
a
C18:2 n6 C18:3 n3
Animal Biotechnology and Genomics Education
Animals can metabolize ALA to form n3 long chain PUFA through a series of desaturation and elongation steps
18:1n-9
18:3n-3
ALA
18:4n-3 20:4n-3 20:5n-3
EPA
20:4n-6
AA
20:3n-618:3n-618:2n-6
LA
FAT-2
FAT-1 FAT-1 FAT-1 FAT-1
18:1n-9
18:3n-3
ALA
18:4n-3 20:4n-3 20:5n-3
EPA
20:4n-6
AA
20:3n-618:3n-618:2n-6
LA
FAT-2
FAT-1 FAT-1 FAT-1 FAT-1
Linoleic Acid
-Linolenic Acid
Arachidonic Acid
Eicosapentaenoic Acid
n-6
n-3
Animal Biotechnology and Genomics Education
In vivo proof of concept using Fat 1 gene - Beth Kao (MS)
Transgenic mice (C57/BL6 x DBA) were generated by pronuclear microinjection of the SalI/NotI fragment fragment cut from the AVE013 vector, containing the C. elegans omega-3 fatty acid desaturase (Fat 1) under the control of the goat beta-casein promoter of the pBC1 mammary expression vector (Invitrogen).
AVE013 (pBC1+FAT1)
22844 bp
Delta15 desaturase
Ampicillin resisitance (bla)
Beta-casein intron 1
Beta-casein intron 7
Beta-casein intron 8
Goat Beta-Casein Promoter
Bla promoter
pBR322-derived origin
TATA box
Beta-casein 3' genomic DNA
Beta-casein exon 1 (untranslated)
Beta-casein exon 2 (partial; untranslated)
Beta-casein exon 7 (partial; untranslated)
Beta-casein exon 8 (untranslated)
Beta-casein exon 9 (untranslated)
2X beta-globin insulator
SalI (14236)
Not I (8359)
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Introducing Hugh....
Hemizygous sire that produced the Fat 1-12 mice – sired over 25 litters to date – he turned 2 on Saturday so we had a party
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Fat 1-12 line had a single insertion site
Southern analysis of the FAT 1-12 transgenic mice. Lane 1: EcoRI digested founder, lane 2: EcoRI digested F2 mouse, lane 3: HindIII digested founder, lane 4: HindIII digested F2 mouse, lane 5: 1kb Extend Ladder (Invitrogen). Banding pattern suggests a tail to tail ( )
orientation of two transgenes in a single insertion site.
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Milking day at the mouse house
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Good transgene expression – full length Fat 1 expression
mouse beta-casein
fat-1 CDS
Goat beta casein 5’ UTR / fat-1 intron spanning primers
Animal Biotechnology and Genomics Education
Milk triacylglycerol fatty acid composition
0
2
4
6
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10
12
14
C18:2n6 C18:3n3 C20:4n6 C20:5n3
Fatty Acids
Mo
l %
Fa
tty A
cid
s
Control
Transgenic
*
***** **
Purina 5015 mouse chow (11% total fat) 28% linoleic acid (18:2n6) 0.7% α-linolenic acid (18:3n3)
Animal Biotechnology and Genomics Education
0
2
4
6
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10
12
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18
20
C18:2n6 C18:3n3 C20:4n6 C20:5n3
Fatty Acids
Mo
l %
Fa
tty A
cid
s
Control
Transgenic**
*** ** ***
Purina 5015 mouse chow (11% total fat) 28% linoleic acid (18:2n6) 0.7% α-linolenic acid (18:3n3)
Milk phospholipid fatty acid composition
Animal Biotechnology and Genomics Education
Fatty Acids of Brain Lipids from 15 Day Old Mice
0
2
4
6
8
10
12
14
16
C18:2n6
C18:3n3
C20:4n6
C22:2 n6
C20:5n3
C22:3 n3
C22:4 n6
C22:5 n6
C22:5n3
C22:6n3
Fatty Acid
Mo
l %
Fatt
y A
cid
Control
Transgenic
*
***
**
**
**
***
*
*
*
n6 DPA n3 DHA
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In mammalian brains, long-chain PUFA begin to accumulate in the brain during the last days of gestation.
The accretion rate of DHA is the most concentrated compared to other fatty acids
The majority of brain growth, or the “brain growth spurt,” begins after birth and takes place during the first few weeks post-parturition
Afterward, the adult brain has been described to tenaciously retain the long-chain PUFA, even during n3 and n6 fatty acid dietary deficiency
Model for infant nutrition ?
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Do mouse pups nursed on Fat1 transgenic dams have increased plasma n3 LC-PUFA levels and higher rates of DHA accretion in the brain ?
• Eight transgenic females and eight nontransgenic full sib control females were placed on a high safflower oil diet (11.5% fat: 69% 18:2n6 LA, 16% oleic acid 18:1, 3% stearic acid 18:0, and 10% palmitic acid 16:0) from breeding to 21 days post-parturition.
• There were 33 pups reared in each treatment group.
• No difference in growth rate was observed.
Animal Biotechnology and Genomics Education
Mouse milk fat is ~ 1 % phospholipid
Fatty Acids of Milk Phospholipids
0
5
10
15
20
25
C18 C18:1
cis9&10
C18:2n6 C18:3n3 C20:4n6 C20:5n3 C22:5 n6 C22:5n3 C22:6n3
Fatty Acids
% M
ol Fatt
y A
cid
Control
Transgenic
***
***
***
*
Safflower mouse chow (11.5% total fat) 69% linoleic acid (18:2n6) 0.168 % -linolenic acid (18:3n3) 0% n6 or n3 PUFA
Animal Biotechnology and Genomics Education
Fatty Acids of Milk Triacylglycerols
0
5
10
15
20
25
C18 C18:1
cis9&10
C18:2n6 C18:3n3 C20:4n6 C20:5n3 C22:5 n6 C22:5n3 C22:6n3
Fatty Acids
% M
ol Fa
tty A
cid
Control
Transgenic
**
*****
Mouse milk fat is ~ 99 % triacylglycerol
No significant difference in n6 DPA or DHA
Animal Biotechnology and Genomics Education
Fatty acids of plasma lipids in 21 day pups nursed on control or transgenic dams
Fatty Acids of Plasma Lipids in 21 Day Old Mice
0.000
5.000
10.000
15.000
20.000
25.000
30.000
C18 C18:1
cis9&10
C18:2n6 C18:3n3 C20:4n6 C20:5n3 C22:5 n6 C22:5n3 C22:6n3
Fatty Acids
% M
ol F
atty
Acid
Control
Transgenic
*****
**
***
Animal Biotechnology and Genomics Education
Brain Lipids of 21 Day Old Mice
0.000
2.000
4.000
6.000
8.000
10.000
12.000
14.000
16.000
C18:2n6 C18:3n3 C20:4n6 C20:5n3 C22:5 n6 C22:5n3 C22:6n3
Fatty Acids
% M
ol F
atty
Acid
Control
Transgenic
***
***
**
***
***
***
Brain lipids of 21 day pups nursed on control or transgenic dams
Animal Biotechnology and Genomics Education
Brain fatty acid time course of pups nursed on control or transgenic dams
0
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6
8
10
12
14
16
Day 0 Day 12 Day 21
% m
ol fa
tty
ac
ids
n6 DPA Control
n6 DPA TG
n3 DHA Control
n3 DHA TG
n3 DHA
n6 DPA
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Conclusions from Fat 1 studies
Nursing transgenic dams increased levels of n3 PUFA in the
plasma and brain of pups
Nursing transgenic dams increased the amount of DHA in the
brain over time despite no significant increase of DHA in milk
Pups nursed on nontransgenic dams possessed only ~50% of
the DHA in the brain at 21 days, and had significantly elevated
levels of n6 docosapentaenoic acid (DPA, 22:5n6) in the plasma
and brain despite no significant difference of n6 DPA in milk
Other studies have found that during n3 PUFA deficiency, neural
DHA composition decreases significantly, and n6 DPA replaces
DHA in the n3 deficient tissues.
Significant DHA deficiency states are associated with decreased
behavioral function in animal models.
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What about Fat 2 ?
18:1n-9
18:3n-3
ALA
18:2n-6 LA
FAT-2
FAT-1
Fat 1 is sufficient in monogastrics
but in ruminants need Fat 2 too
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Control pup raised on Fat 2 dam
Fat 2 pup raised on control dam
10-day old pups – two litters both of 7 babies
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10-day old pups – two litters both of 7 babies – individual weights. Some Fat 2 babies never make it beyond 11-12 days
1 2 3 4 5 6 7
0
2
4
6
8
Grams
Mouse number
2-14 Dam
Control Dam
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FAT2 babies never grow as well as FAT1 or C57 controls
0
2
4
6
8
10
12
10 21
Age (days)
Gra
ms
Fat1-8
fat2-1
fat2-22
fat2-27
Fat2-14 never made it to 21 days
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Other researchers have noted agalactia with a ∆12 desaturase
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Future work
Solve the Fat 2/∆12 desaturase problem
Cross Fat 2 with Fat 1 mice
Obtain In vivo proof of concept
Move into a ruminant
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Conclusion
Increasing the proportion of ω3 fatty acids found in ruminant products offers a way to improve the nutritional content of an important component of the American diet.
The development of sustainable dietary sources of land-based beef and dairy products rich in ω3 polyunsaturated fatty acids would provide a compelling example of how animal biotechnology could be utilized to produce functional foods for the enhancement of human health.
Animal Biotechnology and Genomics Education
Acknowledgements
Alison Van Eenennaam Laboratory, UC Davis – Beth Kao (Fat 1 mouse studies)
– Kristine Lewis (Mouse Colony Management)
Ed DePeters Laboratory, UC Davis
– Scott Taylor (Milk Analysis)
Juan Medrano Laboratory, UC Davis – Kerri Morimoto (In vitro proof of concept in HC11 cells)
John Browse Laboratory, Washington State Institute of Biological Chemistry, Washington State University, Pullman, WA for C. elegans fat-1 and fat-2 cDNA.
Supported by NIH grant 1R03HD047193-01