Dr. Kevin J. HarvatineAssociate Professor of Nutritional PhysiologyDepartment of Animal Science

How Bioactive FA Change Milk Fat Synthesis

The Fatty Acid Forum sponsored by

§Overview of milk fat§Historical Theories of Milk Fat Depression§Mechanism of Bioactive FA in the Mammary Gland

How Bioactive FA Change Milk Fat SynthesisWhat is Milk Fat?

§Lipid droplet surrounded by a phospholipid membrane with “milk fat globular membrane proteins”

§Mostly triglycerides (~95%)§Three sources of milk FA–De novo–Mixed Source (16 Carbon)–Preformed

How Bioactive FA Change Milk Fat SynthesisWhat is Milk Fat?

is a specific inhibition of milk fat yield

How Bioactive FA Change Milk Fat SynthesisWhat is Milk Fat?

“Classical Diet-Induced” Milk Fat Depression

(MFD)

“BiohydrogenationInduced” Milk Fat Depression (MFD)OR

§Up to a 50% decrease in milk fat§Greater reduction in de novo synthesized FA than

preformed FA§No change in milk or milk protein yield§Little change in DMI§Occurs during disrupted rumen fermentation

How Bioactive FA Change Milk Fat SynthesisBiohydrogenation Induced MFD

§Recognized over 150 years ago§Naturally occurs with certain diets–high concentrate, low fiber– low in effective fiber– plant and fish oil supplements– unsaturated fatty acids

§Many theories proposed that fit into two categories1.Substrate Limitation2.Direct Inhibition of Milk Fat Synthesis

§Key insight ~2000 with investigation of conjugated linoleic acids (CLAs)

How Bioactive FA Change Milk Fat SynthesisHistory of Milk Fat Depression

Overall studies provide little or no support for theories involving a shortage of precursors for milk

fat synthesis

§Dietary fat deficiency§Acetate deficiency (¯ rumen production) §b-hydroxybutyrate deficiency (anti-ketogenic effects of

propionate)§Glucogenic-insulin theory (tissue competition)

How Bioactive FA Change Milk Fat SynthesisShortage of Precursors Theories

Normal Diet HG/LF DietMilk yield No change

Milk fat, g/d 683 363

Rumen VFA, %

Acetate 67 46

Propionate 21 46

Butyrate 11 9

Acetate/Propionate 3.2 1.0

From Davis et al. 1967 and Bauman et al. 1971.

How Bioactive FA Change Milk Fat SynthesisAcetate Limitation Theory

Normal diet HG/LF DietRumen Production, moles/d

Acetate 29.4 28.1a

Propionate 13.3 31.0b

B-hydroxybutyrate 7.0 9.1c

aDavis et al. 1967bBauman et al. 1971cPalmquist et al. 1969

How Bioactive FA Change Milk Fat SynthesisBut….Amount of Acetate is What is Important

Acetate infusion also does not rescue Biohydrogenation-Induced Milk Fat Depression

Increased Glucose

Adipose Tissue

Mammary Gland

How Bioactive FA Change Milk Fat SynthesisGlucogenic-Insulin Theory

Increased Insulin

Acetate, BHBA, & Preformed FA

Increased Propionate

High Grain:Forage Diet

-40

-20

0

20

40

60

C6 to C14:1De Novo FA

C16 +C16:1

HG/LF dietPlant oil supplementInsulin clampGlucose infusion

% Ch

ange

How Bioactive FA Change Milk Fat SynthesisChanges in Milk Fat Composition

Biohydrogenation induced MFD is different from glucose and insulin

C18 + C18:1Preformed FA

How Bioactive FA Change Milk Fat SynthesisDirect Inhibition Theory of Milk Fat Depression

Davis and Brown (1970) & Selner & Schultz (1980)

Altered rumen microbes and fermentation

Formation of specific trans fatty acids in the rumen

Inhibition of mammary lipid synthesis enzymes

Milk fat depression

How Bioactive FA Change Milk Fat SynthesisRumen Biohydrogenation

Altered Pathways

Linoleic Acid(cis-9, cis-12 C18:2)

Stearic Acid(C18:0)

Rumenic Acid(cis-9, trans-11 CLA)

Vaccenic Acid(trans-11 C18:1)

Stearic Acid(C18:0)

Alternate CLA Isomers(cis-9, trans-11 CLA)

Trans-C18:1 Isomers(trans-10 C18:1)

Griinari and Bauman, 1999

1.5

2

2.5

3

3.5

-2 -1 1 2 3 4 5 6 7 8

Day

Mil

k Fa

t (p

erce

ntag

e)

Infusion

Control

1.5

2

2.5

3

3.5

-2 -1 1 2 3 4 5 6 7 8

cis-9, trans-11 CLA

trans-10, cis-12 CLA

Baumgard et al., 2000

How Bioactive FA Change Milk Fat SynthesisEffect of CLA isomers on Milk Fat

trans-10 cis-12 CLADecreased Milk Fat

Basal membrane ER- membrane Apical membrane

LPL DGAT

ACC FAS

FA synthesis de novo (C4 - C16)

TAGsynthesis

Glycerol

Glucose

SFA (C16 - C18)

Synthesis Secretion

Acetate

ßHBA

VLDL

TAG

Glycerol

Glucose

Circulation Translocation

UFA

DesaturaseGPAT

FABP

XO

BTN1A1MUC1

ADFP

MFGM

How Bioactive FA Change Milk Fat SynthesisHow is Milk Fat Synthesis Changed?

Biochemical process/enzymeDiet-

induced MFD10,12 CLA-

induced MFDSynthesis de novo

acetyl CoA carboxylasefatty acid synthase

A,B,CA,B,E

CD,E

Preformed fatty acidslipoprotein lipasefatty acyl CoA ligase

A,EA,

A,E

DesaturationD9-desaturase A,C,E D

Esterificationacylglycerol phosphate acyl transferaseglycerol phosphate acyl transferase

AA

DD

Citations are as follows: A = Peterson et al., 2003; B = Piperova et al., 2000;C = Ahnadi et al., 2002; D = Baumgard et al., 2002; E = Harvatine and Bauman, 2006.

How Bioactive FA Change Milk Fat SynthesisLipogenic Genes Are Coordinately Decrease During MFD

RXR

How Bioactive FA Change Milk Fat SynthesisBioactive Sensors

Modified from Duplus-Forest, 2002

Fatty Acids

ChREBP

SREBP1

S14

RXR

ER Stress RXR PPAR

FABP

AMPK

RXR LXR

HNF4α

§SREBP1c– Central regulator of lipid synthesis–Binding element in the promoter of many lipogenic enzymes

§Spot 14- “Lipogenic Factor”–No definitive function–Highly correlated with fat synthesis–Most highly down-regulated by CLA in microarray–Knock-out mouse– 50% decrease in milk fat

How Bioactive FA Change Milk Fat SynthesisCandidate Regulatory Genes

** **

***

***

Harvatine and Bauman, 2006

How Bioactive FA Change Milk Fat SynthesisMammary Gene Expression

*** ***

** **

0

20

40

60

80

100

120

FASN LPL

Rela

tive

mRN

A A

bund

ance

Con CLA LF/HO

0

20

40

60

80

100

120

SREBP1 S14

Rela

tive

mRN

A A

bund

ance

Con CLA LF/HO

Coordinated Decrease in Lipogenic Enzymes

Decrease in Regulators of Lipid Synthesis

§Diet-induced MFD is not caused by a limitation of substrate, although substrate can change milk fat yield to a small degree in some situations (not MFD)

§Bioactive FA cause diet-induced MFD and have provided significant insight into the regulation of mammary lipid synthesis

§SREBP1 and S14 are important signaling factors, but other signaling systems are undoubtedly also involved

How Bioactive FA Change Milk Fat SynthesisConclusions and Future Directions

Daniel Rico, Michel Baldin, L. Whitney Rottman, Mutian Niu, Natalie Urrutia, Isaac Salfer, Andrew Clark, Liying Ma, and Jackie Ying

Collaborators:Dr. Dale Bauman- Cornell UniversityDr. Yves Boisclair- Cornell University

Supported By:National Research Initiative Competitive Grants Program, CSREES, USDA [Grant #2006-35206-16643; PI Bauman]

Agriculture and Food Research Initiative Competitive Grant no. 2010-65206-20723 and 2015-67015-23358 from the USDA National Institute of Food and Agriculture [PI Harvatine]NIH-NICHD Grant Number 5R03HD068661-02 [PI Harvatine]ELANCO Animal Health, Berg and Schmidt, and Novus

How Bioactive FA Change Milk Fat SynthesisAcknowledgements

Daniel Rico, Michel Baldin, L. Whitney Rottman, Mutian Niu, Natalie Urrutia, Isaac Salfer, Andrew Clark, Liying Ma, and Jackie Ying

Collaborators:Dr. Dale Bauman- Cornell UniversityDr. Yves Boisclair- Cornell University

Supported By:National Research Initiative Competitive Grants Program, CSREES, USDA [Grant #2006-35206-16643; PI Bauman]

Agriculture and Food Research Initiative Competitive Grant no. 2010-65206-20723 and 2015-67015-23358 from the USDA National Institute of Food and Agriculture [PI Harvatine]

NIH-NICHD Grant Number 5R03HD068661-02 [PI Harvatine]

ELANCO Animal Health, Berg and Schmidt, and Novus

Thank You!

How Bioactive FA Change Milk Fat SynthesisAcknowledgements

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