THE 5' AMP-ACTIVATED PROTEIN KINASE IN BOVINE MAMMARY EPITHELIAL CELLS

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THE 5' AMP-ACTIVATED PROTEIN KINASE IN BOVINE MAMMARY EPITHELIAL CELLS

By Jianhui Huang

Department of Animal Science McGill University, Montreal, Canada

November 2018

A thesis submitted to McGill University in partial fulfilment of the requirements of the degree of

MASTER OF SCIENCE

© Jianhui Huang, 2018

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Abstract

The synthesis of milk components requires a sufficient supply of energy substrates.

In the ruminant, acetate and glucose provide the main source of energy for the animal. 5' adenosine monophosphate-activated protein kinase (AMPK) is a cellular energy gauge that controls anabolic and catabolic processes to maintain a balance between energy supply and demand. The overall objective of this study was to assess the role of AMPK on de novo lipid synthesis and glucose metabolism in response to energy substrates in bovine mammary epithelial cells (BMEC).

Primary BMEC were isolated from lactating mammary tissue of 3 independent cows. The cells were induced to differentiate by incubation in Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12 (DMEM/F12) supplemented with lactogenic hormones (5 µg/mL each of insulin, prolactin and hydrocortisone) for 4 d. Site- specific phosphorylation was measured by immunoblotting. The expression of lipogenic and glycolytic genes was quantified by qPCR. The quantification of de novo lipid synthesis and lactose synthesis was measured by the incorporation of

3H-acetate and ¹⁴C-glucose into cellular lipids and lactose, respectively. Data were analyzed by ANOVA using a randomized complete block design with PROC MIXED in SAS. Treatment differences were considered significant when P < 0.05.

We first characterized activation of AMPK by pharmacological compounds in BMEC. Cells were treated with 100 μM A-769662, an allosteric activator of AMPK, or vehicle control for 16 h. Interestingly, while treatment of BMEC cells A-769662 had no effect on AMPK phosphorylation for 16 h, it remarkably increased phosphorylation of its targets, ACC and TSC2, by 144% and 26% (P < 0.05). The role of AMPK in de novo lipid synthesis and glucose metabolism was subsequently assessed. Activation of AMPK by A-769662 resulted in increasing gene expression of SREBF1 and SCD1 by 42% and 63%, respectively. In addition, AMPK reduced the proportion of mature SREBP-1c by 19%. These changes resulted in a decrease of 19% in the incorporation of acetate into total cellular lipid. Activation of AMPK also increased expression of VEGF-A by 46%, but did not change the expression of SLC2A1, which encodes for GLUT1. Lactose synthesis was reduced

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by 24%. To assess the regulation of AMPK by energy substrate availability in BMEC, cells were incubated in control medium containing 4 mM D-glucose and 1 mM sodium acetate, or medium lacking glucose or acetate for 4 h. Deprivation of glucose or acetate significantly promoted phosphorylation of AMPKα at Thr172 by 84% or 58%, respectively, compared to cells in control medium. To define the sensitivity of AMPK to glucose levels, BMEC were treated incubated in DMEM media without or with 1, 2 or 4 mM glucose for 4 h. The phosphorylation of AMPKα, as well as ACC and TSC2, differed from 4 mM change with completely devoid of glucose for 4 h. We conclude that senses energy substrate deprivation AMPK in BMEC. Pharmacological activation of AMPK inhibits de novo lipid and lactose synthesis through transcriptional and post-translational mechanisms in BMEC.

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RÉSUMÉ

La synthèse des composantes du lait nécessite un apport suffisant en substrats énergétiques. Chez le ruminant, l’acétate et le glucose constituent les principales sources d’énergie pour l’animal. La protéine kinase activée par l'adénosine monophosphate (AMPK) est une jauge d'énergie cellulaire qui contrôle les processus anaboliques et cataboliques afin de maintenir un équilibre entre l'offre et la demande en énergie. L'objectif général de cette étude était d'évaluer le rôle de l'AMPK sur la synthèse de novo des lipides et sur le métabolisme du glucose en réponse à des substrats énergétiques dans les cellules épithéliales mammaires bovines (BMEC). Les BMEC primaires ont été isolées à partir de tissu mammaire en lactation de 3 vaches indépendantes. Les cellules ont été induites à se différencier par incubation dans du Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12 (DMEM / F12) additionné d'hormones lactogènes (5 µg / mL d'insuline, de prolactine et d'hydrocortisone) pendant 4 jours. La phosphorylation spécifique au site a été mesurée par immunoblot. L'expression de gènes lipogéniques et glycolytiques a été quantifiée par qPCR. La quantification de la synthèse de novo des lipides et du lactose a été mesurée par l'incorporation de 3H-acétate et de 14C-glucose dans les lipides cellulaires et le lactose, respectivement. Les données ont été analysées par ANOVA en utilisant une conception de bloc complète randomisée avec PROC MIXED dans SAS. Les différences de traitement ont été considérées comme significatives lorsque P

<0,05. Nous avons d'abord caractérisé l'activation de l'AMPK par des composés pharmacologiques dans le BMEC. Les cellules ont été traitées avec 100 pM de A- 769662, un activateur allostérique de AMPK, ou un contrôle pendant 16 h. Fait intéressant, bien que le traitement des cellules BMEC A-769662 n’ait pas d’effet sur la phosphorylation de l’AMPK pendant 16 h, il a remarquablement augmenté la phosphorylation de ses cibles, ACC et TSC2, de 144% et 26% (P <0,05). Le rôle de l'AMPK dans la synthèse de novo des lipides et le métabolisme du glucose a ensuite été évalué. L'activation de l'AMPK par l'A-769662 a entraîné une augmentation de l'expression des gènes de SREBF1 et SCD1 de 42% et 63%,

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respectivement. De plus, l'AMPK a réduit de 19% la proportion de SREBP-1c mature. Ces changements ont entraîné une diminution de 19% de l'incorporation d'acétate dans les lipides cellulaires totaux. L'activation de l'AMPK a également augmenté l'expression de VEGF-A de 46%, mais n'a pas modifié l'expression de SLC2A1, qui code pour GLUT1. La synthèse de lactose a été réduite de 24%. Pour évaluer la régulation de l'AMPK par la disponibilité du substrat énergétique dans le BMEC, les cellules ont été incubées dans un milieu de contrôle contenant du D- glucose 4 mM et de l'acétate de sodium 1 mM, ou du manque de glucose ou d'acétate pendant 4 h. La privation de glucose ou d'acétate a favorisé de manière significative la phosphorylation de l'AMPKα, au niveau de la Thr172, de 84% ou 58%, respectivement, par rapport aux cellules du milieu témoin. Pour définir la sensibilité de l'AMPK aux taux de glucose, les cellules BMEC ont été traitées et incubées dans un milieu DMEM sans ou avec du glucose à 1, 2 ou 4 mM pendant 4 h. La phosphorylation de l'AMPKα, ainsi que de l'ACC et du TSC2, différait de 4 mM avec une absence totale de glucose pendant 4 h. Nous concluons que la détection de la privation de substrat énergétique AMPK dans BMEC. L'activation pharmacologique de l'AMPK inhibe la synthèse de novo des lipides et du lactose par le biais de mécanismes transcriptionnels et post-traductionnels dans les cellules BMEC.

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Acknowledgements

I would like to express my gratitude to my advisor and mentor, Dr. Sergio Burgos.

You have fostered a learning environment that ensures graduate student success.

I also thank you for giving me the opportunity to get involved in this community. My time at McGill has been a rewarding experience and I am deeply grateful for both your patience and guidance. I thank the members of my advisory committee, Dr.

Humberto Monardes and Dr. David Zadworny, for their continued support throughout the MSc program. Special thanks to Marc-Antoine Guesthier, Stefanie LaForce, Wei Si for assisting me with laboratory procedures and listening to my stories. To my close friends, too many to list, thank you for giving me the chance to share my frustrations whether in the laboratory or in my life over a beer at the Maple. The personal financial support from Marian and Ralph Sketch Fellowship is gratefully acknowledged. The research financial support for this project was provided by NSERC. This thesis is dedicated to my parents and my sister, Huaichang Huang, Xianlan Huang and Zhiyan Huang. I am grateful for their continued love, support and guidance.

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Table of Contents

Abstract ... i

Acknowledgements ... v

Table of Contents ... vi

Glossary of Abbreviations ... ix

List of Figures ... xii

1. Literature Review ... 1

1.1 Milk Synthesis and Secretion ... 1

1.1.1 Milk Lactose ... 2

1.1.2 Milk Lipids ... 4

1.1.3 Milk Proteins ... 12

1.2. AMPK ... 16

1.2.1 AMPK Structure ... 16

1.2.2 Mechanism of Activation... 20

1.3 Role of AMPK in regulation of milk synthesis ... 22

1.3.1 Lipid metabolism ... 23

1.3.2 Glucose metabolism ... 25

1.3.3 Energy metabolism ... 27

1.3.4 Protein metabolism ... 29

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2. Manuscript ... 32

Role of AMP-activated protein kinase on lipid synthesis and glucose metabolism in bovine mammary epithelial cells ... 32

2.1 Introduction ... 33

2.2 Materials and Methods ... 34

2.2.1 Materials ... 34

2.2.2 Isolation and culture of bovine mammary epithelial cells ... 35

2.2.3 Cell treatments ... 36

2.2.4 Lipid synthesis assay ... 37

2.2.5 Lactose synthesis assay... 37

2.2.6 Immunoblotting ... 37

2.2.7 Subcellular fractionation ... 38

2.2.8 RNA extraction and quantitative real-time PCR ... 39

2.2.9 Statistical analysis ... 39

2.3. Results ... 40

2.3.1 Effect of A-769662 on phosphorylation of AMPK, TSC2 and ACC in BMEC ... 40

2.3.2 Effect of A-769662 on de novo lipid synthesis and lipogenic gene expression in BMEC ... 40

2.3.3 Effect of A-769662 on SREBP-1c protein abundance, proteolytic processing and subcellular localization in BMEC ... 41

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2.3.4 Effect of A-769662 on lactose synthesis and glycolytic gene expression in BMEC ... 42 2.3.5 Effect of glucose and acetate deprivation on AMPK activity in BMEC . 42 2.4. Discussion ... 43 2.5 Conclusions ... 46 4. References ... 64

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Glossary of Abbreviations

4EBP1 Eukaryotic translation initiation factor 4E-binding protein 1

ACC Acetyl-CoA carboxylase

ADP Adenosine diphosphate

AGPAT 1-acylglycerol-3-phosphate acyltransferase AICAR 5-aminoimidazole-4-carboxamide riboside

AID Auto-inhibitory domain

AKT PKB Protein kinase B

AMP Monophosphate

AMPK AMP-activated protein kinase

AMPKK AMPK kinases

ATGL Adipose triglyceride lipase

ATP Adenosine triphosphate

BHBA ß-hydroxybutyrate

BMEC Bovine mammary epithelial cell

CaMKKβ Ca2+/calmodulin-dependent kinase kinase β

CBM Carbohydrate-binding module

CBS Cystathionine β-synthase

CD36 Cluster of differentiation 36

ChREBP Carbohydrate-responsive element-binding protein CLA Conjugated linoleic acid

CPT1 Carnitine palmitoyl transferase 1

CTD C-terminus domain

DGAT Diglyceride acyltransferase DHAP Dihydroxyacetone phosphate DMSO Dimethyl sulfoxide

DNA Deoxyribonucleic acid

EAA Essential amino acid

eEF2 Eukaryotic elongation factor 2

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eEF2K Eukaryotic elongation factor 2 kinase eIF4E Eukaryotic translation initiation factor 4E

ER Endoplasmic reticulum

F1,6BP Fructose 1,6-bisphosphate FABP Fatty acid binding protein

FAS Fatty acid synthase

G6P Glucose 6-phosphate

GADP Glyceraldehyde 3-phosphate GLUT Glucose transporter

GPAT Glycerol-3-phosphate acyltransferase GSK3β Glycogen synthase kinase 3 beta HBSS Hank’s balanced salt solution

HK Hexokinase

HMGCR 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase HSL Hormone-sensitive lipase

INSIG Insulin-induced gene

IRS1 Insulin receptor substrate-1

JAK Janus kinase

KD Kinase domain

LCFA Long-chain fatty acid

LKB1 Liver kinase B1

LXR Liver-X receptor

LXRE LXR response element

MAFbx Muscle atrophy F-box

MCA Malonyl-CoA

mRNA Messenger ribonucleic acid mtGPAT Mitochondrial GPAT

mTORC Mechanistic target of rapamycin complex MuRF1 Muscle RING-finger protein-1

NADH Reduced nicotinamide adenine dinucleotide

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NADPH Reduced nicotinamide adenine dinucleotide phosphate NEAA Non-essential amino acid

nSREBP Nuclear SREBP

p70S6K Ribosomal protein S6 kinase beta-1

PA Phosphatidic acid

PDH Pyruvate dehydrogenase

PFK2 Phosphofructokinase 2

PI3K Phosphoinositide 3-kinase

PPAR Peroxisome proliferator-activated receptor

PPP Pentose phosphate pathway

PRL Prolactin

qPCR Quantitative real-time PCR RER Rough endoplasmic reticulum Rheb Ras homolog enriched in brain

rpS6 Ribosomal protein S6

SCAP SREBP cleavage activated protein

SCD Stearoyl-CoA desaturase

SGLT Sodium-glucose co-transporter

SREBP Sterol regulatory element-binding protein

STAT Signal transducers and activators of transcription TAK1 Transforming growth factor-β-activated kinase 1 TBS-T Tris-buffered saline with Tween 20

TCA Tricarboxylic acid

TG Triglyceride

tRNA Transfer RNA

TSC2 Tuberous sclerosis complex 2 α-RIM Regulatory subunit interacting motif

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List of Figures

Figure 1.1. The upstream signal pathway of SREBP-1c ... 11

Figure 1.2. Schematic of AMPK subunits and domain structure ... 18

Figure 1.3. The activation mechanism of AMPK ... 22

Figure 1.4. The regulation of AMPK ... 23

Figure 2.1. Effect of A-769662 on phosphorylation of AMPK, TSC2 and ACC in BMEC ... 48

Figure 2.2. Effect of A-769662 on de novo lipid synthesis in BMEC. ... 49

Figure 2.3. Effect of A-769662 on lipogenic gene expression in BMEC ... 52

Figure 2.4. Effect of A-769662 on SREBP-1c protein abundance, proteolytic processing and subcellular localization ... 54

Figure 2.5. Effect of A-769662 on lactose synthesis in BMEC. ... 55

Figure 2.6. Effect of A-769662 on glycolytic gene expression in BMEC... 57

Figure 2.7. Effect of deprivation of energy substrates on phosphorylation of AMPK, TSC2 and ACC in BMEC. ... 59

Figure 2.8. Dose response of glucose on phosphorylation of AMPK, TSC2 and ACC in BMEC ... 61

Figure 2.9. Time course of deprivation of glucose on phosphorylation of AMPK, TSC2 and ACC in BMEC ... 63

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1. Literature Review

1.1 Milk Synthesis and Secretion

The major constituents in bovine milk are water, proteins, lipids, lactose, minerals and vitamins. They are either synthesized in the mammary gland from blood-borne precursors or transferred directly from the general circulation. The majority of milk protein and fat as well as all of the lactose are synthesized by bovine mammary epithelial cells (BMEC); immunoglobulins, minerals and vitamins are mainly taken up from the blood through the basal membrane of mammary epithelial cells through their respective transport systems. In addition to the synthesis of milk components, BMECs secrete milk components through the apical membrane into the lumen of the alveoli through different secretory pathways. Milk protein, lactose, calcium and phosphorus are secreted by secretory vesicles (Linzell and Peaker, 1971). Milk fat is secreted in the form of milk fat globules, in which triglycerides (TG) are surrounded by a protein membrane (Peterson et al., 2004). Amino acids, glucose and most minerals get in and out of the cell through specific transporters.

Finally, a paracellular pathway mediates the transport of other small molecules between mammary epithelial cells and the milk (Shennan and Peaker, 2000). The synthesis and secretion of milk components have been considered as energy- consuming processes that requires energy and the participation of corresponding enzymes. The energy uptake of the mammary gland is equal to the sum of the milk energy output and the energy consumed by the mammary gland. Based on the calculations by Na et al. (2014), the ratio of milk energy output to energy uptake of the mammary gland is 0.88.

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1.1.1.1 Lactose Synthesis

Lactose, a disaccharide composed of one molecule of glucose and one molecule of galactose, is the main carbohydrate in milk. Lactose is the main osmole in milk and it is an important determinant of milk volume, and thereby the percentage composition of other milk components by drawing water into the mammary epithelial cells (Xiao and Cant, 2005). To synthesize lactose, glucose is first converted to galactose in the cytoplasm. The goat mammary gland is estimated to utilize up to 85% of the glucose taken up from the circulation for lactose synthesis (Annison and Linzell, 1964). Glucose and galactose are then transported into the Golgi apparatus for further processing. The lactose synthesis reaction is catalyzed by lactose synthase, an enzymatic complex composed of the mammary-specific milk protein α-lactalbumin and β-1, 4-galactosyltransferase in the Golgi compartment (Keenan et al., 1972). A large number of studies has been extensively conducted to investigate the effect of glucose infusions on lactation performance and metabolic profiles. Hurtaud et al. (2000) found that although 1,500 g/d duodenal glucose infusions significantly improved milk yield and tended to reduce milk fat content, they had no significant effect on milk lactose content, suggesting the extra glucose supply may provide an energy source rather than lactose precursor to the bovine mammary gland. However, other experiments have shown inconsistent results (Huhtanen et al., 2002, Rigout et al., 2002).

1.1.1.2 Glucose Uptake

Glucose uptake is considered to be the rate-limiting step for lactose synthesis. The glucose uptake is conducted by glucose transporters - a class of functional proteins mediating the movement of glucose across the cell membrane. Glucose transporters are classified in two major families based on transport characteristics:

glucose transporter (GLUT) and sodium-glucose co-transporter (SGLT).

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The most studied glucose transporter in the mammary gland is GLUT1, a 54 kDa protein composed of 492 amino acids. The GLUT1 protein is relatively highly expressed in both lactating and non-lactating bovine mammary tissue and is considered to mediate the majority of glucose uptake during lactation (Zhao et al., 1996). Heard et al. (2000) summarized the process of glucose uptake in human erythrocytes into three major steps: (1) firstly, translocation of glucose under the mediation of GLUT1, (2) then, release of glucose into an internal transporter space that was formed by allosteric binding of intracellular adenosine triphosphate (ATP) on cytoplasmic domain of the GLUT1 proteins under the cell membrane (3) lastly, release of glucose from the space into cytosol for further processing. They also found asymmetry of the glucose transporters would be lost if cellular ATP was depleted, which caused incomplete function of glucose transporters, suggesting energy supply played an important role in the glucose uptake of human erythrocytes. Xiao and Cant (2003) demonstrated that GLUT1 in BMEC had a similar homotetrameric pattern as that described in erythrocytes.

The tissue and subcellular distribution of glucose transporters in the bovine mammary gland has been studied in detail. Zhao et al. (1993) detected the expression of GLUT1, 3, 4, 5 transcripts and the inexistence of GLUT2 in the lactating bovine mammary gland, by Northern blotting analysis, and that lactating mammary glands express a high level of GLUT1 and a low level of GLUT3, 4, 5.

However, Komatsu et al. (2005) subsequently found that the expression of GLUT4 was also not detected in the lactating bovine mammary gland. The presence of two novel glucose transporters, GLUT12 and GLUT8, were later identified. These studies revealed that GLUT8 has the second highest level of expression in the bovine mammary gland (Zhao and Keating, 2007). Although SGLT1 and SGLT2 were also detected in both the lactating or non-lactating bovine mammary gland, their expression is low (Zhao, 2014) and their physiological role in lactation remains to be elucidated.

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Due to the involvement of glucose uptake in the process of lactose synthesis, activated glucose transporters are accompanied by lactose synthesis. In humans, GLUT1 is the major transporter responsible for glucose transport on the membrane of mammary epithelial cells and the Golgi apparatus; galactose is transported into the site of lactose synthesis by both SGLT1 and SGLT2 (Lin et al., 2016). Glucose passes across the basolateral membrane of BMECs and Golgi membrane through GLUT1, GLUT8 and SGLT1; however the uptake of galactose into the Golgi lumen is predominantly facilitated by GLUT1 (Zhao, 2014).

In addition to glucose transporters, the process of glucose uptake is also found to be regulated by the endocrine system. Prolactin, the key lactogenic hormone, has been found to stimulate the uptake of 2-deoxy-D-glucose by the mammary tissue (Peters and Rillema, 1992). Injection of exogenous growth hormone also enhanced glucose uptake by the mammary gland without changing plasma glucose concentration in cows (Davis et al., 1988) or the glucose concentration in goats milk (Faulkner, 1999). These observations suggest that these hormones may stimulate glucose transport into the mammary gland. However, the use of exogenous growth hormone for 63 days did not alter the messenger ribonucleic acid (mRNA) expression of GLUT1 in the lactating bovine mammary gland (Zhao et al., 1996). Therefore, Tanwattana et al. (2003) proposed that exogenous growth hormone may enhance glucose uptake by increasing blood flow to the mammary gland rather than induction of GLUT1 expression in BMECs.

1.1.2 Milk Lipids

1. 1.2.1 De novo fatty acid synthesis

Milk lipids are primarily composed of TGs, which accounts for more than 95% of total milk lipids. The remaining milk lipids include are diglycerides (2%), phospholipids (1%), cholesterol (0.5%) and free fatty acids (0.1%) (Jensen et al., 1991). There are at least 400 different fatty acids presented in milk (Lindmark Månsson, 2008). The milk TGs are composed of three fatty acids esterified to a

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glycerol backbone. Different kinds of fatty acids are acquired from different sources.

Almost all short chain (4-8 carbons) and medium chain (10-14 carbons) fatty acids and about half of 16-carbon fatty acids are synthesized within the mammary gland, the other half of 16-carbon fatty acids and long-chain fatty acids (LCFA) (greater than 16 carbons) are obtained directly from the blood (Bauman and Griinari, 2003).

The mechanisms of de novo fatty acid synthesis have several marked differences between ruminant and non-ruminant species. In ruminants, acetate and butyrate are the main precursors for de novo fatty acid synthesis. These volatile fatty acids originate from the fermentation products of carbohydrates in rumen metabolism.

Butyrate is converted into ß-hydroxybutyrate (BHBA) by the rumen epithelium, entering the fatty acid synthesis pathway as a primer only, providing the first four carbons for about 50% of de novo synthesized fatty acids. BHBA also can be converted into 2 acetyl-CoA molecules in the mitochondria; however, these are retained in the mitochondria and cannot be used as substrates for fatty acid synthesis (Hurley, 2009). The major source of reduced nicotinamide adenine dinucleotide phosphate (NADPH) required for de novo fatty acid synthesis is provided by the non-oxidative glucose metabolism of the pentose phosphate pathway, 2 NADPH molecules are produced from each molecule of glucose that enters this pathway (Champe et al., 2005).

De novo fatty acid synthesis is a complex metabolic pathway in the cytoplasm, and there are several major enzymes that regulate this process, including acetyl-CoA carboxylase (ACC), fatty acid synthase (FAS) and stearoyl-CoA desaturase (SCD).

Acetyl-CoA, the main substrate for fatty acid synthesis, is primarily produced by the pyruvate dehydrogenase (PDH) reaction in the tricarboxylic acid (TCA) cycle and by fatty acid oxidation (King, 1996). The first step of fatty acid synthesis is the generation of malonyl-CoA (MCA), in which ACC catalyzes the combination of bicarbonate (HCO3–) and acetyl-CoA to synthesize MCA. MCA and acetyl-CoA subsequently go into the MCA pathway, which through condensation, reduction, dehydration reactions, and another reduction step under the action of FAS causes

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the addition of two carbons elongating the fatty acid chain. The MCA pathway is then repeated until the fatty acid has reached a certain chain length for acyl thioesterase to cleave off from the carrier protein (Chan and Vogel, 2010). SCD1 is responsible for the conversion of C16:0 - C18:0 fatty acids into corresponding monounsaturated fatty acids and conjugated linoleic acids (CLA) in the mammary gland of ruminants (Corl et al., 2001), playing a crucial role in unsaturated fatty acid synthesis.

1.1.2.2 Preformed Fatty Acids

In addition to de novo synthesis, a portion of fatty acids in milk are also derived from preformed fatty acids. These include about half of fatty acids with 16 carbon and all of those with longer carbons chains. Duncan and Garton (1963) detected that the main hemic preformed fatty acids were C16:0, C18:0 and C18:1 in ruminants; they are derived from dietary lipids or endogenous catabolism of TGs.

Moate et al. (2008) found that a high level of fish oil fatty acids in the diet led to a decrease in the production of all the major individual preformed fatty acids with the exception of C20:5 and C22:6 in the bovine milk. Therefore, the preformed fatty acid is highly related to nutrient composition.

The uptake of free fatty acid from plasma is regulated by cluster of differentiation 36 (CD36) and fatty acid binding protein (FABP). CD36, also known as fatty acid translocase, functions as a receptor of exogenous fatty acid, suggesting it plays a critical role in fatty acid translocation. The role of CD36 in fatty acid translocation has been observed in several cell types, including rat adipocyte, heart and skeletal muscle cells, with highest expression in adipocyte and mammary epithelial cells (Xu et al., 2013). The up-regulated expression of FABP and CD36 during lactation and the down-regulated expression during involution have been observed in the bovine mammary gland (Spitsberg et al., 1995), showing their relationships with physiological variations, which further influences lipid metabolism. Barber et al.

(1997) proposed the cooperation of intracellular FABP and CD36 in the transportation of fatty acids across the cell membrane. The mammary gland is

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considered to be actively involved in the uptake of LCFA. Abumrad et al. (1993) found that CD36 existed in the membrane of milk fat globules and was regulated by lactogenic hormones. Yonezawa et al. (2004) revealed that the treatment with exogenous LCFA significantly elevated the mRNA expression of CD36, TG secretion and lipid droplets in primary BMECs, demonstrating that a positive effect of CD36 on the uptake of LCFA in BMEC. Xu et al. (2013) proposed that CD36 enhanced the rate of exogenous fatty acid uptake by improving intracellular esterification efficiency rather than strengthening the transport effect across the plasma membrane in HEK293 cells, which may indicate a similar mechanism is present in BMEC.

FABPs are responsible for intracellular transport of LCFA (Weisiger, 2002). Ten FABPs have been found in mammals, including FABP1-9 and FABP12 (Yamamoto et al., 2009). They are highly conserved proteins with a molecular weight of 14-15 kDa (Smathers and Petersen, 2011). The expression of FABP is tissue-specific, and mRNA of all FABP isoforms, except FABP2, were present in bovine mammary tissue (Storch and Thumser, 2010). In BMEC, FABP3 expression is reduced due to posttranscriptional gene silencing of sterol regulatory element-binding protein 1 (SREBP-1c), suggesting that FABP3 may be a downstream target of SREBP-1 (Ma and Corl, 2012). Bionaz and Loor (2008a) found that greater abundance and up-regulation of FABP3 mRNA, which has a high affinity with stearic and palmitic acid, in the lactating bovine mammary gland (Whetstone et al., 1986). These findings indicate that FABP3 may play an important role in milk fat synthesis.

1.1.2.3 Triglyceride Synthesis

TGs are synthesized in the smooth endoplasmic reticulum of mammary epithelial cells. De novo synthesized and exogenous fatty acids are activated by addition of acyl-CoA and then esterified to glycerol-3-phosphate to generate TGs. The glycerol used for TG synthesis is mainly derived from glucose in BMECs. The newly synthesized TGs form small lipid droplets that fuse together and move

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toward the apical membrane, which are eventually released to the lumen of the alveolus as a milk fat globule. Several enzymes have been identified as critically important for the regulation of TG synthesis by BMEC including: glycerol-3- phosphate acyltransferase (GPAT), 1-acylglycerol-3-phosphate acyltransferase (AGPAT), lipin and diglyceride acyltransferase (DGAT) (Coleman and Lee, 2004, Bionaz and Loor, 2008a). AGPAT and lipin are the most abundant isoforms in the bovine mammary gland (Bionaz and Loor, 2008a). GPAT catalyzes the esterification of the first acyl-CoA to the sn-1 position of glycerol-3-phosphate to form lysophosphatidic acid. AGPAT catalyzes esterification of the second acyl- CoA to the sn-2 site to synthesize phosphatidic acid (PA). Lipin is PA phosphatase, converting PA into diglyceride. Finally, DGAT1, which is present in the membrane of ER, acylates the sn-3 site of the glycerol molecule to complete TG synthesis.

Although DGAT is a rate-limiting enzyme for the synthesis of TGs in rat hepatocytes (Mayorek et al., 1989), Bionaz and Loor (2008a) proposed that DGAT1 plays a minor role in the milk fat synthesis compared to other genes in TG synthesis. However, the addition of sodium acetate in culture medium enhance the expression of DGAT in BMECs, which may be caused by the inhibition effect on the AMP-activated protein kinase (AMPK) (Na et al., 2014).

1.1.2.4 Regulation of milk fat synthesis

A large number of factors have been found to affect enzymes involved in fatty acid synthesis. Recent findings reported by Zhang et al. (2014b) indicated that methionine (Met) upregulated the mRNA expression of ACC, FAS and SCD, as well as TG secretion in BMECs by activating the mechanistic target of rapamycin complex (mTORC1)/ribosomal protein S6 kinase beta-1 (S6K1, known as p70S6K) signaling pathway and enhancing glycogen synthase kinase 3 beta (GSK3β) phosphorylation. In addition, incubation of BMECs with sodium acetate, sodium butyrate or β-sodium hydroxylate also increases the expression of ACC and FAS, as well as TG secretion, while the addition of sodium caprylate shows an inhibition on the expression of ACC, FAS and SCD1 (Kong et al., 2012). Zhang et al. (2014a)

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tested the activity of those enzymes in lactating Holstein cows that are classified based on different milk fat content, showing that the ACC activity was not significantly different between dairy cows with high fat content in milk and those with low fat content in milk, while the activity of FAS and SCD was markedly higher in cows with high milk fat. Therefore, energy substrates and nutrient availability can have regulatory effects on the expression and activity of several key enzymes in de novo fatty acid synthesis in the bovine mammary gland. The contribution of nutrient-sensing pathway to regulate milk lipid synthesis remains to be further studied.

Numerous mechanisms have been proposed by which nutrient and energy substrate availability regulate milk fat synthesis either directly or indirectly.

SREBPs, consisting of two subtypes: SREBP-1 and SREBP-2, are considered to be key transcriptional factors for regulation of lipid metabolism in response to nutrients (Brown and Goldstein, 1997). SREBP-1, including SREBP-1a and SREBP-1c isoforms, preferentially regulate the expression of genes involved in fatty acid synthesis; whereas SREBP-2 mainly regulated the synthesis and uptake of cholesterol (Edwards et al., 2000, Moon et al., 2001). Overexpression of SREBP-1c was found to cause disorders of lipid metabolism, resulting in accumulation of excessive lipid in non-fat tissues (Unger and Zhou, 2001).

SREBPs are initially attached to the membrane of the ER. In order to enter the nucleus to conduct their transcriptional regulatory functions, the precursor form of SREBPs has to be converted into active mature nuclear SREBP (nSREBP) by proteolysis. This process is regulated by cholesterol-sensing protein SREBP cleavage activated protein (SCAP) and the ER-resident membrane protein, insulin- induced gene (INSIG). SCAP forms a complex with SREBP that can bind reversibly to INSIG. Low intracellular sterol levels induce SCAP-SREBP complex dissociation from INSIG and binding to coat protein II, which transports the complex to the Golgi apparatus. After two cleavages by site 1 and site 2 protease, the SREBP precursor releases an active N-terminal portion that enters the nucleus where it binds to the sterol regulatory element sequence, activating transcription

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of downstream target genes. Thus, increasing intracellular sterol binds to INSIG and SCAP, causing the SREBP-SCAP complex to remain in the ER (Brown and Goldstein, 1997, Goldstein et al., 2002).

Figure 1.1 shows signal pathways upstream of SREBP-1c. Both mTORC1 and AMPK were found to regulate the expression of SREBP-1c. The studies conducted by Inoki et al. (2003) and Tee et al. (2003) demonstrated that insulin enhanced mTORC1 activation through the phosphatidylinositol-4,5-bisphosphate 3- kinase/protein kinase B (AKT) signaling pathway. They found that AKT phosphorylated tuberous sclerosis complex 2 (TSC2), an upstream negative regulator of mTORC1, leading to inactivation of Ras homolog enriched in brain (Rheb), a direct activator of mTORC1. Owen et al. (2012) found that low level of rapamycin, an allosteric inhibitor of mTORC1, suppressed transcription of SREBP- 1, suggesting the importance of mTOR regulation on SREBP-1. In addition, mTORC1 also can activate S6K1 to enhance the proteolytic cleavage on SREBP- 1. Around the same time, mTORC1 was shown to enhance nuclear SREBP protein abundance by inhibiting lipin1 (Peterson et al., 2011). Therefore, mTORC1 can regulate SREBP by controlling transcription and proteolytic cleavage of SREBP-1, as well as the abundance of nSREBP-1. AMPK, activated by metformin, was found to suppress SREBP-1c cleavage and nuclear translocation, as well as repress SREBP-1c target gene expression in hepatocytes, leading to reduced lipogenesis and lipid accumulation (Li et al., 2011).

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Figure 1.1. The upstream signal pathway of SREBP-1c

Liver-X receptors (LXR), LXRα and LXRβ, are nuclear factors that regulate SREBP-1 transcription. Two LXR response elements (LXREs) are present in the promoter region of SREBP-1c, indicating SREBP-1c may regulated by LXR (Yoshikawa et al., 2001). Indeed, LXR dramatically enhanced the transcription of SREBP-1c, resulting in higher fatty acid synthesis, even in the presence of high level of intracellular sterol (Ye and DeBose-Boyd, 2011). In contrast, PUFA was found to inhibit the combination of LXR with LXRE on the SREBP-1c, leading to lower SREBP-1c RNA expression and less fatty acid synthesis (Ou et al., 2001).

There has been intensive research interest in the role of LXR in milk fat synthesis during lactation. Rudolph et al. (2007) showed that both LXRα and LXRβ were highly expressed in lactating mice. The expression of LXRα in the mammary gland in early pregnancy was about 10 times higher than that in the early lactation of rodents (Anderson et al., 2007). However, Farke et al. (2008) reported the expression of LXRα mRNA was not different between lactating and dry cows.

Acute and chronic treatment of BMECs with T0901317, an LXR agonist, dramatically increased de novo fatty acid synthesis (McFadden and Corl, 2010).

This effect was associated with upregulation of transcription, translation and

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proteolytic cleavage of SREBP-1 in response to LXR activation, which further led to increase the mRNA abundance of FAS. Thus, SREBP-1 upregulated by LXR activation may contribute to fatty acid synthesis in BMECs.

Similar to LXR, peroxisome proliferator-activated receptor gamma (PPARγ) functions as a nutrient-sensitive transcription factor, and could be an important regulator of SREBP-1. Bionaz and Loor (2008a) measured the changes in the relative mRNA abundance of SREBPs and PPARγ during lactating in the bovine mammary gland; there was a significant increase in the expression of PPARγ mRNA in the lactating mammary gland. Incubation of BMEC with rosiglitazone (a PPARγ agonist) up-regulates the expression of FAS and SREBP-1c (Qin et al., 2008). The specific role and mechanism for PPARγ regulation on SREBP-1c in the bovine mammary gland is yet to be determined.

The expression of SREBP-1c in the mammary gland during lactation is significantly up-regulated in mice and cows (Anderson et al., 2007, Rincon et al., 2012). Barber et al. (2003) reported that SREBP-1c was the major regulator of de novo fatty acid synthesis in sheep. Harvatine and Bauman (2006) found that there was a reduction of SREBP-1c and SREBP-1c-related enzymes in dairy cows that have the diet- induced decrease in milk fat synthesis, and trans-10, cis-12 CLA significantly reduced the mRNA expression of SREBP regulatory proteins, INSIG1 and INSIG2 (Harvatine and Bauman, 2006).

1.1.3 Milk Proteins

1.1.3.1 Amino Acid Uptake

Amino acids are the constituents of proteins. From a nutritional perspective, amino acids can be classified as essential amino acid (EAA) or non-essential amino acid (NEAA). Mammary epithelial cells take up free amino acids and small peptides from plasma as precursors for protein synthesis. Indeed, more than 90% of the milk proteins arise from free amino acids and small peptides in the mammary gland

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of dairy cows (Davis, 1976, Backwell et al., 1996). Mepham et al. (1992) proposed that the concentration of milk component precursors in blood, the blood flow to the mammary gland, and the transport of precursors into mammary epithelial cells were important factors that influenced synthesis of milk components. Free amino acids are taken up by mammary epithelial cells through amino acid transport systems at the cell membrane, but there is a lack of information on how small peptides enter mammary epithelial cells.

The findings reported by Lapierre et al. (2012) and Apelo et al. (2014) indicated that the uptake of mammary EAA played a crucial role in the study of lactation in dairy cows. Mepham (1982) classified EAA into two groups based on the uptake to output ratio by the mammary gland. Met, phenylalanine, tyrosine (Tyr) and tryptophan were considered as group 1, because uptake by the mammary gland was similar to secretion in milk protein, which suggested that they were mainly used for synthesis of milk proteins in the mammary gland; group 2 included arginine (Arg), isoleucine (Ile), leucine (Leu), valine, threonine (Thr) and lysine (Lys), where uptake exceeded milk protein output, therefore, they may have different purposes in addition to the synthesis of milk proteins. Zamwel et al. (2002) found that proline uptake was less than the amount in milk protein, histidine (His) and alanine were greater than those in milk protein; unlike the result from Mepham (1982), Thr and Lys were considered as group 1 in the mammary gland of dairy cows. Bequette et al. (1998) proposed that branched-chain amino acids with excessive mammary uptake may use for oxidation in the mammary gland. Apelo et al. (2014) detected that maximizing the uptake of EAA by regulating diet and increasing the efficiency of amino acid utilization by the mammary gland resulted in higher efficiency of dietary N utilization in dairy cows; they also suggested research priorities in milk protein synthesis should mainly focus on the metabolic utilization of EAA rather than metabolic protein.

14 1.1.3.2 Protein Synthesis

The major proteins in milk are mammary-specific casein proteins (α, β, γ and κ), lactoglobulin and lactalbumin, which account for more than 90% of milk protein. As with other proteins, milk proteins are synthesized from amino acids that are activated by ATP and acylated to their cognate transfer RNA (tRNA) in the cytoplasm. mRNAs encoding specific milk protein localize to the ribosomes of rough endoplasmic reticulum (RER). Based on the recognition between anticodons in tRNA and the codons in mRNA, amino acids release from tRNA and join the polypeptide chain, further forming milk proteins. Newly synthesized milk proteins are continually processed and modified in the RER, for example, by glycosylation of kappa-casein. After that, they are transferred from the RER to the Golgi apparatus for phosphorylation. The Golgi apparatus integrates casein molecules, calcium and phosphorus into casein micelles that are then transported to the apical membrane via secretory vesicles sprouted from Golgi apparatus. The vesicles fuse with the apical membrane and release proteins into the alveolar lumen.

The main factors influencing the rate of milk protein synthesis are nutrient availability and hormone levels. These cues are relayed by signaling pathways.

The Janus kinase/signal transducer and activator of transcription (JAK/STAT) signaling pathway mainly regulates the synthesis of casein; whereas the mTOR signaling pathway has an important role in amino acid sensing and metabolism.

They are subject to internal regulators and external environmental factors, such as nutrients, hormones and temperature regulation.

The JAK/STAT5 signaling pathway plays a key role in the casein synthesis in the mammary gland with the regulation of prolactin (PRLR). PRLR regulates the morphological and biochemical differentiation of the epithelial cells during pregnancy and the milk protein synthesis in the mammary gland during lactation (Groner, 2002). JAK2 is a non-receptor cytoplasmic protein Tyr kinase involved in the intracellular domain of PRLR, and STAT5 is a deoxyribonucleic acid (DNA)

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binding protein. The binding of PRL to its receptor initiates the dimerization of the receptor and activates JAK2, leading to phosphorylation of multiple Tyr residues of PRLR and STAT proteins (Radhakrishnan et al., 2012). Phosphorylated STAT5 separates from the receptor and moves into the nucleus where it binds to DNA and modulates the activity of target-related genes, such as the β-casein gene (Kazansky et al., 1995). With the exception of PRL, growth hormones, epidermal growth factors, erythropoietin, other cytokines and hormones have been reported to activate STAT protein in different tissues or stages. Specific amino acids has been reported to regulate milk protein synthesis through the expression of STAT5a gene in the JAK2/STAT5 signaling pathway (Liu et al., 2012).

Besides the association of the JAK2/STAT5 signaling pathway with milk protein synthesis, mTOR is also an important signaling pathway for amino acid metabolism in the mammary gland. It perceives amino acid abundance, and stimulates milk protein production via phosphoinositide 3-kinase (PI3K)-AKT- mTOR pathway (Hay and Sonenberg, 2004). Components of the mTOR pathway can sense the absence of several specific amino acids, and the sensitivity changes with different amino acids. Leu and Arg are considered to be highly involved in mTORC1 activation. Studies have shown that leu, Arg and His play an important role in the regulation of casein gene translation and the expression of genes related to the mTOR pathway (Wang et al., 2014, Gao et al., 2015). The effect of amino acids is interacting in the mTOR pathway. The addition of both Ile and Thr to BMECs can affect the phosphorylation of mTOR, but their roles are opposite, Ile promotes the phosphorylation of ribosomal protein S6 (rpS6), while Thr inhibits the effect (Apelo et al., 2014). It has been reported that Rag GTPase is essential for mTORC1 activation by Leu, while mTORC1 activation via glutamine stimulation is dependent on the activity of vacuolar-type H⁺-ATPase in lysosomes and vacuoles, the mechanism is different from the way of Leu (Jewell et al., 2015), showing that the mechanism of mTOR activation by amino acids is more complex. Appuhamy et al. (2011) found that EAA enhanced the phosphorylation of mTOR, S6K1, eukaryotic translation initiation factor 4E-binding protein 1 (4EBP1) and insulin

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receptor substrate-1 (IRS1) on the mTOR signalling pathway; therefore, protein synthesis enhanced by amino acid nutrition could be associated with phosphorylation of mTOR and its downstream signals 4EBP1 and S6K1.

1.2. AMPK

1.2.1 AMPK Structure

AMPK is a sensor of cellular energy supply and consumption in eukaryotes. It plays a vital role in the regulation of cellular energy homeostasis and nutrient metabolism in response to energy availability. In normal physiological conditions, cells keep a high concentration of ATP in order to maintain basic metabolic activity. However, conditions of energy shortage caused by a reduction of ATP production or an increase of ATP consumption, or both, result in an increase in the AMP/ATP ratio, leading to activation of AMPK.

Mammalian AMPK was independently identified by two groups in 1973. Beg et al.

(1973) found that 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase (HMGCR), a key enzyme in the cholesterol metabolic pathway, was inactivated via a cytoplasmic component in the presence of Mg²⁺ and ATP. This inactivation was later shown to be accompanied by phosphorylation of HMGCR (Brown et al., 1975).

In the same year, Carlson and Kim (1973a) reported that ACC, a key enzyme in the fatty acid metabolic pathway, was phosphorylated and then inactivated by a protein kinase. It was not definitively proved that the kinase, which phosphorylated ACC, was activated by AMP until 1980 (Yeh et al., 1980). Subsequent studies revealed that the same kinase, whose activity is regulated by AMP, is responsible for phosphorylation of HMGCR and ACC (Carling et al., 1987).

Figure 1.2 shows the schematic of AMPK subunits and domain structure. As a heterotrimeric protein complex, an integral AMPK complex is made up of one

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catalytic subunit (α) and two regulatory subunits (β and γ) (Hardie, 2016). The bovine genome encodes two isoforms for α and β subunits, and three isoforms for γ subunit. Accordingly, there are 12 possible distinct combinations of αβγ complexes (Herzig and Shaw, 2017). The actual composition of the heterotrimeric AMPK complex in a given tissue is determined by the expression level of the different subunit isoforms, suggesting that some combinations may have different functions (Salt and Hardie, 2017). In dairy cows, skeletal muscle has the highest mRNA expression level of AMPKα subunits compared to adipose, liver and mammary tissues; α2 isoform is predominantly expressed in heart, skeletal muscle and liver, while α1 isoform tissue distribution is ubiquitous (McFadden and Corl, 2009). Thornton et al. (1998) found the β1 isoform was mainly distributed in rat liver and brain, but there was lower expression present in the kidneys and skeletal muscle; rat skeletal muscle had the greatest abundance of β2 subunit, whereas there was low level expression in the kidneys, liver and lungs. The mRNA level of γ subunit is highly expressed across a range of rat tissues, including heart, lung, skeletal muscle, liver, and kidney (Stapleton et al., 1996), whereas γ1 complex comprises more than 80% of the activity in rat liver, lungs, heart, skeletal muscle, kidneys and pancreas (Cheung et al., 2000). Different subcellular localizations for isoform within cells have also been reported. For example, the α1 isoform localizes in the nonnuclear fraction, whereas the α2 isoform has a wide localization in the rat hepatocyte (Witczak et al., 2008), which may cause a differential pattern of the AMPK activity within cells.

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Figure 1.2. Schematic of AMPK subunits and domain structure 1.2.1.1 AMPK α subunit

The α subunit of AMPK consists of four functional domains, which includes a kinase domain (KD) at the N-terminus, an auto-inhibitory domain (AID), an α-linker and a C-terminus domain (CTD). The KD is constituted by a small N-lobe and a large C-lobe, which is followed by a small globular domain, the AID. The activity of the KD is tenfold higher than that of the KD-AID complex, suggesting AID suppresses the activity of the KD (Hardie et al., 2016). The α-linker, between AID and CTD, is associated to γ subunit through two regulatory subunit interacting motifs (α-RIM). The binding of adenosine monophosphate (AMP) to γ subunit leads the α-linker to pull then AID away from KD, thus preventing its inhibitory effect on KD (Li et al., 2015).

There are several phosphorylation sites on AMPKα subunit, some of them are known to have critical roles in AMPK function. Hawley et al. (1996) identified Thr172, in the activation loop of KD, as the major site phosphorylated by AMPKK on AMPK. Willows et al. (2017) also suggested that Thr172 was a critical phosphorylation site for maximal activity of AMPK in mammalian cells. They tested AMPK activities in the presence of AMP and 991 that activated AMPK through γ and β subunits respectively, rather than Thr172 site, finding non-phosphorylated Thr172 AMPK was much less active than the Thr172 phosphorylated AMPK; cells

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which are knockout two major upstream kinases of AMPK, including Ca²⁺/calmodulin-dependent kinase β (CaMKKβ) and liver kinase B1 (LKB1), by using CRISPR-mediated genome editing, fail to detect Thr172 phosphorylation of AMPK and its activity. Therefore, the activation of AMPK by energy stress and calcium level change is accounted for Thr172 phosphorylation. In addition to Thr172, Thr258 and serine (Ser) 485 on the α subunit are found to be phosphorylated by several kinases. Ser485 can be autophosphorylated in response to energy depletion and other regulators to limit AMPK activation. AKT phosphorylates Ser485 in response to insulin stimulation, and protein kinase A phosphorylates Ser485 to blunt AMPK activity (Hurley et al., 2006), suggesting that phosphorylation of Ser485 may also play a role in AMPK regulation in vivo. Protein kinase A also phosphorylates Ser173 to preclude the Thr172 phosphorylation by LKB1 and ultimately inhibits the activation of AMPK (Djouder et al.).

1.2.1.2. AMPK β subunit

In eukaryotes, the β subunit of AMPK is comprised of a carbohydrate-binding module (CBM) and a CTD. The CBM is a central conserved region that allows AMPK to bind to glycogen particles, thereby referred to as glycogen binding domain. The CBM in β2 isoform is presented to have a higher affinity for glycogen than that in β1 isoform (Koay et al., 2010). The CTD is responsible for interaction with the with the γ subunit and the N-lobe of the KD on the α subunit, acting as a scaffold of the heterotrimeric complex. The mutagenesis of phosphorylation on Ser108 is found to have a positive effect on the reactivity of AMPK with its allosteric activator, AMP (Warden et al., 2001).

1.2.1.3 AMPK γ subunit

The γ subunit contains two tandem Bateman domains, each domain is constituted by two cystathionine β-synthase repeats (CBS) (Hardie et al., 2016). The CBSs are responsible for binding to regulatory adenine nucleotides AMP, adenosine diphosphate (ADP) and ATP (Li et al., 2015). The assembly of four CBSs provides

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four potential ligand-binding clefts; three of them are capable of binding with AMP, the remaining one is always unoccupied (Li et al., 2015), which caused by the lack of the conserved aspartate residues on CBS2, that assists CBS to bind with the ribose ring of adenine nucleotides (Salt and Hardie, 2017). Previous study shows the activation of AMPK via binding of AMP on the AMPKγ subunit has a thousand- fold higher than that by upstream AMPK kinases (AMPKK) (Zhao et al., 2017).

1.2.2 Mechanism of Activation

1.2.2.1 Phosphorylation of Thr172 on the α subunit

The known mechanism of activation of AMPK are shown in Figure 1.3. There are a variety of pathways to modulate the activity of AMPK. As a sensor of cellular energy status, AMPK regulates a variety of metabolic processes. Under conditions of high intracellular AMP concentration, which results in an increase in AMP/ATP ratio, AMP binds the γ subunit of AMPK thereby activating the kinase activity of the complex. Once activated, AMPK phosphorylates downstream targets to stimulate ATP production while switching off the metabolic pathways that consume ATP to conserve energy. The increase of AMP/ATP ratio is highly related to the physiological conditions, such as glucose deprivation, hypoxia, ischaemia and heat shock (Hardie, 2011a). Four mechanisms for the binding of AMP on the γ subunit to activate AMPK have been proposed: 1) AMP stimulates the phosphorylation of Thr172; 2) AMP prevents Thr172 from the dephosphorylation by phosphatases; 3) AMP triggers the activity of AMPK upstream kinases; 4) AMP binding to the γ subunit renders AMPK as a better substrate for upstream kinases (Hawley et al., 1995, Herzig and Shaw, 2017). As ATP competes with AMP for binding to the CBS domains on the γ subunit, the key signal of AMPK activation is the increase of AMP and the reduction of ATP. Therefore, AMPK is regulated by AMP/ATP ratio rather than the respective nucleotide levels.

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Several pharmacological compounds that activate AMPK, either directly or indirectly, have been used to study the function of this protein kinase in cellular functions. According to the classification by Kim et al. (2016), AMPK activators can be divided into different activating mechanisms; modulators that causes AMP or calcium accumulation are classified as indirect activators, such as α-lipoic acid, polyphenols, thiazolidinedione and biguanides, but direct AMPK activators, such as A-769662, activate AMPK by inducing a conformational change rather than altering cellular nucleotide levels. The phosphorylation of Thr172 on AMPK enhances the activity of AMPK by at least 100 times, while allosteric activation of AMPK only increases the activity by five times (Scott et al., 2002). From a quantitative point of view, activation by Thr172 phosphorylation could be more important than the allosteric activation of AMPK.

1.2.2.2 Phosphorylation by 3 upstream kinases

As mentioned earlier, LKB1, CaMKK and transforming growth factor-β-activated kinase 1 (TAK1) are major kinases that directly phosphorylate Thr172 on its α subunit to activate the AMPK complex. LKB1 is thought to play a dominant role, especially when the AMPK activation is induced by energy stress (Herzig and Shaw, 2017). LKB1 was first implicated as a kinase of AMPK in a study of Peutz- Jegher syndrome, as a tumor suppressing gene in human cells (Shaw et al., 2004).

The study by Shaw et al. (2004) revealed that the phosphorylation of Thr172 and the activation of AMPK were not responded to the physiological stimulation and other regulators at all in the LKB1-deficient embryonic fibroblasts of mice; while AMPK activity was recovered after re-introduction of LKB1. Hawley et al. (2003) also found that AMPK was not activated by 5-aminoimidazole-4-carboxamide riboside (AICAR) and metformin in the cell devoid of the LKB1 expression, but which was rescued by the transfection of LKB1. Accordingly, LKB1 is considered a master kinase of AMPK. In addition to LKB1, Hawley et al. (2005) showed that CaMKK phosphorylates Thr172 site in AMPKα in response to an increase of intracellular Ca²⁺ flux, without alteration of the AMP/ATP ratio. However, unlike

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LKB1, CaMKK is only expressed in a limited number of tissue cells, in which the isoforms in the nervous system and vascular endothelial cells are mainly CaMKKα and CaMKKβ, respectively. Finally, TAK1 and TAK1-binding protein 1 were also shown to phosphorylate activate AMPK by phosphorylation at Thr172 (Momcilovic et al., 2006), but the physiological relevance of this event is not known.

Figure 1.3. The activation mechanism of AMPK

1.3 Role of AMPK in regulation of milk synthesis

The downstream targets of AMPK are highly related to the regulation of energy and nutrient metabolism. The regulation of metabolic pathway by AMPK, including lipid, protein and glucose, was previously reported through directly modifying the activity of a range of specific enzymes and including HMGCR, ACC, hormone- sensitive lipase (HSL), and glycogen synthase in a variety of cell types. As described in Figure 1.4., AMPK inhibits multiple biosynthetic pathways involved in ATP-consuming cellular processes, including synthesis of glycogen, protein, lipid, cholesterol and gluconeogenesis; and opens the several catabolic pathways

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involved in ATP-generating cellular activities, such as uptake and oxidation of glucose, glycolysis, fatty acid oxidation and mitochondrial biosynthesis.

Figure 1.4. The regulation of AMPK

1.3.1 Lipid metabolism

1.3.1.1 Fatty acid oxidation and synthesis

Previous studies demonstrated that induction of AMPK activity reduces the rate of fatty acid synthesis and speeds up fatty acid oxidation via inhibitory phosphorylation of ACC (Merrill et al., 1997, Hardie and Pan, 2002). The It has been shown to decrease ACC phosphorylation in mice with muscle-specific AMPKα2 in the muscle (Chen et al., 2015). ACC is a specific enzyme catalyzing the irreversible carboxylation of acetyl-CoA to produce MCA in the fatty acid synthesis pathway (Hardie, 2011b). In mammals, ACC has two isoforms: ACC1 and ACC2; the main difference is that ACC1 lacks the N-terminal mitochondrial targeting segment. As the first product in the step of lipid synthesis pathway, MCA is also a strong inhibitor of carnitine palmitoyl transferase 1 (CPT1), a rate-limiting enzyme that promotes the import and oxidation of LCFA into mitochondria. CPT1 activity is regulated by the negative feedback of MCA, thereby controlling the fatty acid oxidation in mitochondria. When activated AMPK phosphorylates and down- regulates Ser79 on the ACC, the decrease of ACC activity reduces the production

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of MCA, which further weakens the inhibitory effect on the activity of CPT1, thereby, the rate-controlling step in β-oxidation increases and the lipid synthesis slows down (McGarry et al., 1977). Thus, in response to low cellular energy supply, AMPK activation enhances the flux of acyl-CoA for β-oxidation pathway rather than the TG synthesis pathway to reduce ATP utilization and promote ATP generation.

In bovine hepatocytes, adiponectin is found to regulate lipid metabolism through activating AMPK (Ref). Phosphorylated AMPKα activates PPARα to elevate the expression of its target genes, thereby enhancing lipid oxidation, and simultaneously supresses SREBP-1c and carbohydrate-responsive element- binding protein (ChREBP) to reduce the expression of their target genes involved in lipid synthesis. Due to the combined effects on lipid metabolism, the level of TG significantly reduces in adiponectin-treated hepatocytes (Chen et al., 2013). AMPK upregulates the expression of PPARα and PPARδ under prolonged conditions of AICAR treatment, then consequently increases the number of mitochondria and promotes the fatty acid oxidation process, but fatty acid oxidation is repressed when AICAR is treated for a short time (Gaidhu et al., 2011), suggesting the effect of AMPK on the fatty acid oxidation process may be related to the activation period.

1.3.1.2 Lipogenesis and lipolysis

AMPK activated by AICAR in rat hepatocytes was found to decrease the incorporation of radiolabeled oleate and glycerol into TG by 50% and 38%, respectively (Muoio et al., 1999). Li et al. (2013) also demonstrated that adding acetic acid to cultured hepatocytes for 3 h, which activates AMPK, decreased TG synthesis in a dose-dependent manner in bovine hepatocytes. AMPK controls lipid synthesis through downregulating GPAT, which is the lipogenic enzyme catalyzing the first step in de novo TG synthesis and required for milk fat production. In rat hepatocytes, purified recombinant AMPKα supressed mitochondrial GPAT in both time and ATP-dependent manner, and the activity of mitochondrial GPAT is decreased by 22–34% in response to AICAR, but there is no effect on the activity of microsomal GPAT, DGAT or acyl-CoA synthetase (Muoio et al., 1999), which

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may suggest that the AMPK regulates TG synthesis primarily through mitochondrial GPAT (mtGPAT) (Coleman and Lee, 2004). McFadden and Corl (2009) also observed a significant decrease in the mRNA expression of GPAT in response to AICAR in BMEC, in concert with a decrease in fatty acid synthesis.

Taken together, these data suggest that the action of AMPK simultaneously inhibits mtGPAT activity and TG synthesis.

Lipolysis is the process by which lipids are hydrolyzed to release glycerol and free fatty acids. HSL and adipose triglyceride lipase (ATGL) catalyse the rate-limiting step involved in the hydrolyzation of diglyceride and TG, respectively, to free fatty acids. On the one hand, AMPK enhances the activity of ATGL by Ser406 phosphorylation; on the other hand, AMPK allows Ser565 phosphorylation for preclusion of HSL (Zechner et al., 2017). In adipocytes, induction of AMPK activity by AICAR was shown to block isoproterenol-induced HSL translocation to the lipid droplet, which is a necessary process for lipolysis (Daval et al., 2006). Gaidhu et al. (2011) found that the rate of lipolysis in the adipocyte was increased only when mice cells were incubated with AICAR for a prolonged period, and the rate was much slower if AICAR was used in a short time. Accordingly, as with the fatty acid oxidation, the influence of AMPK on lipolysis process may depend on the activation period.

1.3.2 Glucose metabolism 1.3.2.1 Glucose uptake

Glucose is not only an important energy source for the mammary gland, but also a substrate for lactose synthesis. Glucose uptake is a rate-limiting step in the synthesis of lactose (Kronfeld, 1982). It is primarily mediated by GLUTs (Delaquis et al., 1993). As a polar molecule, glucose cannot diffuse freely through the cell membrane due to the hydrophobic nature of the lipid bilayer. Thus, mammary cells

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must rely on a class of glucose carrier proteins distributed on the surface of their cell membranes, GLUTs mediate the passive transport of the extracellular glucose into the cells by facilitated diffusion.

The GLUT family is composed of a variety of transporter proteins that differ widely on their transport characteristics, tissue distribution and subcellular localization.

GLUT1 is the predominant glucose transporter in the mammary gland of rats and dairy cows, but GLUTs 3, 4, 5, 8 and 12 are also detected at a low level in the bovine mammary gland (Macheda et al., 2003, Zhao and Keating, 2007).

Immunohistochemical studies revealed that GLUT1 and GLUT12 was found in the basolateral and apical plasma membrane, respectively, in lactating rat mammary epithelial cells. GLUT1 was also present in the Golgi apparatus during late pregnancy and lactation, suggesting GLUT1 plays an important role in translocation of glucose to Golgi apparatus for lactose synthesis (Macheda et al., 2003).

The regulation of glucose uptake is considered to be a potential mechanism that regulation of lactose synthesis and energy metabolism in the mammary gland. In skeletal muscle, activation of AMPK by stimuli such as exercise induces the expression and translocation of GLUT4 from glucose storage vesicles in the cytoplasm to the cell surface, which leads to an up-regulation of glucose uptake (Russell et al., 1999). However, the expression of GLUT4 is found to decrease from pregnancy to lactation in the rat mammary gland (Burnol et al., 1990).

Activated AMPK is known to participate in the up-regulation of GLUT1, GLUT4 and GLUT8 in equine and mouse skeletal muscle cells (Fryer et al., 2002, de Laat et al., 2015). However, there is limited information regarding the role of AMPK on glucose uptake in the mammary gland. The study of Zhang et al. (2011) showed that glucose uptake and mRNA abundance of GLUT1 dramatically elevated after incubation with AICAR in goat mammary epithelial cells. Since increasing serotonin production elevates AMPK phosphorylation, mRNA abundance and distribution of GLUTs within the rat mammary gland are of a higher level, Laporta

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et al. (2013) hypothesized that the increase of GLUT1 and GLUT8 in the mammary epithelium could be regulated by AMPK. All these changes show the activation of AMPK increases the availability of glucose uptake to meet energy needs and nutrient synthesis. Hence, AMPK could be involved in the regulation of glucose intake in the mammary gland.

1.3.3 Energy metabolism 1.3.3.1 Glycolysis

In the mammary gland, 55-70% of glucose flux to lactose synthesis, most of the remaining glucose is utilized for ATP production through glycolysis and TCA cycle (Guinard-Flament et al., 2006). The process of glycolysis is divided into two separate phases. Upon entry into the cells, glucose is rapidly phosphorylated into glucose 6-phosphate (G6P) by hexokinases (HK) in the cytosol. G6P can then either be used for the direct generation of ATP by glycolysis and the TCA cycle or, alternatively, be directed towards pentose phosphate shunt. The pentose phosphate pathway (PPP) plays a vital role in generation of reducing equivalents, in the form of NADPH, for de novo fatty acid synthesis in the mammary gland of ruminant animals (Aschenbach et al., 2010). Within the first phrase of glycolysis, G6P is further converted to fructose 1,6-bisphosphate (F1,6BP) with consuming two equivalents of ATP. In the second phase F1,6BP is firstly transformed into glyceraldehyde 3-phosphate (GADP) or dihydroxyacetone phosphate (DHAP), which is degraded to pyruvate and is converted into glycerol 3-phosphate through the reduction by glycerol-3-phosphate dehydrogenase for the TG and phospholipid synthesis, respectively (Harding et al., 1975). Finally, four equivalents of ATP and two equivalents of reduced nicotinamide adenine dinucleotide (NADH) are produced to support metabolic activities within the cells, and pyruvate is actively transported across the inner mitochondrial membrane to take part in the TCA cycle in the mitochondrial matrix to produce energy.

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As a cellular energy gauge, AMPK has an important role in regulating ATP production by glycolysis. AMPKα2 subunit has been shown to have a more important role in the regulation of glycolysis than AMPKα1 based on studies in rat muscle cells form knockout mice (Liang et al., 2013). In the skeletal muscle cells, AMPK inhibits glycogen synthase and stimulates the uptake of glucose, thereby promoting glucose oxidation (Halse et al., 2003), consistent with the results in the goat mammary epithelial cells after AICAR treatment (Zhang et al., 2011). Holmes et al. (1999) found that the activity of HK increased by 2.5-fold in rat skeletal muscle tissue under chronic activation of AMPK during 5 consecutive days of AICAR injection, thus making more G6P available for glycolysis and PPP. Two of the important control points in the glycolytic pathway, phosphofructokinase 2 (PFK2) and pyruvate kinase, are also regulated by AMPK. AMPK phosphorylate PFK2 at Ser466, thereby facilitating its activation (Bertrand et al., 2006). Despite the clear importance of AMPK in regulation of glycolytic flux in a variety of cells types and the variability of glucose supply to the mammary gland, the role of AMPK in regulation of glucose uptake and utilization by BMEC has not been characterized.

1.3.3.2 TCA cycle

The TCA cycle is critically important for ATP production, providing the coenzymes, NADH, for de novo fatty acid synthesis, which are derived by oxidation of isocitrate, α-ketoglutarate and L-malate. The regulation of TCA cycle activity is primarily mediated by substrate availability and enzyme activity which controls the rate of flux through the cycle (King, 1996). Pyruvate, the final product in glycolysis, is converted into acetyl-CoA to serve as substrate for the TCA cycle and fatty acid synthesis (Akers, 2016). Winder et al. (2000) found that, with AICAR treatment, citrate synthase, an enzyme controlling one of the flux-determining steps of the TCA cycle, catalyzing the condensation reaction of acetyl-CoA and oxaloacetate into the citrate, showed a significant increase with AMPK activation in white quadriceps and soleus but not in red quadriceps of rat; other two pace-making enzymes in the citric acid cycle, succinate dehydrogenase, catalyzing the oxidation