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Running Head

Running Head: Hepatic gene expression in the periparturient and postpartum period

Hepatic gene expression in the periparturient and postpartum period in dairy cows:
A review

R.C. Doyle
Teagasc, Animal & Grassland Research and Innovation Centre, Moorepark, Fermoy, Co. Cork, Ireland, P61 C996
Division of Animal Sciences, University of Missouri, Columbia, USA 65211

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Corresponding author: [email protected]

ABSTRACT: Research into the physiological and genetic changes from the periparturient to postpartum period has rapidly increased in the last number of years especially in lactating dairy cows. The liver plays a key role in the regulation and coordination of lipid metabolism and is a key component of a cow’s ability to adapt to the onset of lactation. Further research is necessary to investigate how the liver is impacted by genetic selection during all stages of the production cycle and to determine whether perturbations of the liver persist and how they can affect future production.
Key Words: transition period, metabolism, somatotropic axis,
INTRODUCTION
The transition period of a dairy cow is defined as the period from 3 weeks antepartum to 3 weeks postpartum, (Drackley, 1999) is characterized by numerous changes in physiology, energy, metabolism, health and production (Drackley, 1999; Gross et al., 2011a; Gross et al., 2011b). This also the stage of the production cycle where the majority of health disorders such as ketosis, fatty liver, milk fever, retained foetal membranes and displaced abomasum occur. The liver undergoes key adaptations during this period to facilitate the onset of lactation through key processes such as gluconeogenesis and fatty acid oxidation (Greenfield et al., 2000a). These adaptations are regulated by factors including; gene expression changes, nutrient availability and hormonal environment (Loor et al., 2005). The major genes involved in the key functions of the liver can be found in Table.1.
LACTATION INDUCED SOMATOTROPIC AXIS CHANGES
Growth hormone (GH) is a key hormone in the regulation of lactation in dairy cows (Bauman, 1992), its actions are mediated by the GH receptor (GHR) which following binding of the ligand dimerizes and activates the Janus kinase 2 second messenger pathway (Carter-Su et al., 1996). Key functions of GH include increased gluconeogenesis to meet lactogenesis requirements (Knapp et al., 1992) and insulin like growth factor-1 (IGF-1) synthesis and secretion with gene transcription positively regulated by GH (Hannon et al., 1991; Le Roith et al., 2001). Jak2 protein can then phosphorylate signal transducer and activator of transcription (STAT5B) a DNA binding protein which following translocation to the nucleus binds to interferon-gamma-activated sites (GAS) increasing transcription (Lucy et al., 2001). GH receptor mRNA is expressed in two forms 1A and 1B (Adams, 1995) with the adult bovine liver expressing both forms while all other adult tissues only express 1B mRNA, the greatest amount of 1B is found in the liver with 1A being liver specific (Lucy et al., 1998). GH is responsible for stimulating a wide variety of genes and key physiological processes including IGF-1 release from liver (Bichell et al., 1992), lipogenesis inhibition in adipose tissue (Etherton et al., 1987) and muscle growth (Florini et al., 1996). In the periparturient period plasma concentrations of GH increase (Bell, 1995). GH affects multiple tissues but is essential in coordinating nutrient partitioning within the liver (Bauman and Currie, 1980) as GH stimulates lipolysis increasing NEFA concentrations in blood. These NEFA’s can be incorporated into milk fat production or oxidized within the liver. GH is essential in stimulating an increase in liver gluconeogenesis to meet the requirements of lactation (Bell, 1995).
The actions of GH are mediated by GHR present on target cells and the action of GHR second messenger systems (Xu et al., 1995), the amount of GHR mRNA is developmentally, hormonally and nutritionally regulated (Lucy et al., 2001). GHR is located on chromosome 20 in dairy cattle and is encoded by a single gene (Parsons et al., 1998). Three promoters are involved in the control of GHR mRNA UTR 5′ transcription variants, GHR promoter 1 and GHR 1A, GHR P2 and P3 control GHR 1B and 1C. GHR 1A is found within the liver and makes up the majority of GHR mRNA (Jiang and Lucy, 2001b; Lucy et al., 2001). At the time of calving GHR 1A in the liver decreases coinciding with the period of liver refractoriness to GH resulting in decreased GH dependent IGF-1 synthesis (Vicini et al., 1991; Lucy et al., 2001). GHR 1A transcription determines the abundance of GHR in the liver mediating the effects of GH. GHR P1 which transcribes GHR 1A responds to physiological changes in periparturient dairy cows and is independent of GHR P1 and P2.
The physiological and nutritional mechanisms responsible for the down regulation of GHR 1A in the transition period are unknown, however; protein or energy undernutrition can cause a decrease in GHR expression in the liver (Breier, 1999). Stage of lactation also has a significant effect on GHR 1A mRNA with early and mid-lactation cows having less GHR 1A mRNA than late lactation animals (Kobayashi et al., 1999). Expression of GHR 1A and IGF-1 mRNA return to periparturient levels 3 weeks after calving (Radcliff et al., 2003). Expression levels of hepatic IGF-1 mRNA are closely correlated with plasma IGF-1 (Fenwick et al., 2008). The decline in GHR abundance in the periparturient liver is partly attributed to reduced GHR 1A during this time (Kim et al., 2004). Postpartum recoupling of the GH axis is correlated with GHR 1A mRNA and GHR protein and DMI partially controls expression in early postpartum dairy cows (Radcliff et al., 2006). Feed restriction in early lactation reduces GHR abundance in the liver through attenuation of translation rather than decreased GHR 1A abundance (Rhoads et al., 2007).
The actions of IGF-1 are mitigated by the changes in IGF-1 binding proteins (IGFBP’s) in circulation (Jones and Clemmons, 1995). Over 90% of IGF-1 produced by the liver in circulation is bound in a ternary complex with IGBP3 and the acid labile subunit (ALS) (Wathes, 2012). Similar to IGF-1 their production is under the control of GHR. The reduced concentration of IGFBP’s in circulation following calving reduced the half-life of circulating IGF-1 postpartum. In postpartum cows hepatic mRNA expression of IGFBP2 is positively correlated with circulating NEFA and BHB and negatively correlated with liver glycogen, plasma glucose and IGF-1 (Fenwick et al., 2008).
Two weeks prior to parturition feed intake begins to decline in dairy cows with a significant decline observed the day prior and of calving which may contribute to the decrease in liver GHR 1A and IGF-1 synthesis seen in the postpartum period. Key changes in hormones such as progesterone, estradiol as well as the refractoriness of the liver to GH and decreases in HNF-4 may initiate GHR 1A mRNA decrease seen in the transition period (Lucy et al., 2001). The liver expresses the greatest amount of GHR, IGF-1 and IGFBP-2 compared to reproductive tissues including the corpus luteum, dominant follicle or uterus (Rhoads et al., 2008).
TRANSCRIPTION FACTORS AND HEPATIC GENE EXPRESSION
Liver specific transcription factors have the potential to interact with DNA elements within the GHR P1 resulting in the transcription from exon 1 in the liver. Within GHR P1 binding sites for liver-enriched transcription factors CCAAT/enhancer binding proteins (C/EBP) ? and ? and hepatocyte nuclear factor-4 (HNF-4) exist (Lucy et al., 2001). These factors have the potential to regulate specific gene promoters within the liver (De Simone and Cortese, 1992). Binding of HNF-4? and chicken ovalbumin transcription factor II (COUP-TFII) and transcription factors activates GHR P1 (Jiang and Lucy, 2001a; Xu et al., 2004). Increased or over expression of HNF-4 has been shown to increase the activity of GHR P1 in various cell lines with HNF-4 showing to be enriched within the bovine liver (Jiang and Lucy, 2001a). The study also concluded that HNF-4 binding site is conserved across species in the GHR 1P supporting its involvement in the expression of liver specific GHR (Jiang and Lucy 2001). HNF-4 is also expressed within the kidney suggesting it solely is not responsible for the control of GHR 1A expression. Studies in the rat liver have shown that a endotoxin lipopolysaccharide caused a reduction in HNF-4 and GHR abundance (Defalque et al., 1999). Decreased expression of GHR 1A at parturition in the liver has been associated with increased mRNA abundance of HNF-4? and no changes in expression of HNF-4? and COUP-TFII (Jiang et al., 2005). At parturition HNF-4 abundance remains constant indicating a deficit in HNF-4 alone is not responsible for the decreased GHR 1A expression seen in periparturient dairy cows (Kim et al., 2004).
ENERGY BALANCE AND GENE EXPRESSION
The principal challenge facing dairy cows during the transition period is the abrupt and marked increase in nutrient requirements for lactation while dry matter intake (DMI) lags behind. Nutritional and energy requirements can increased 4 fold within 1 day in high yielding dairy cows (Carriquiry et al., 2009). Dairy cows experience a period of negative energy balance during the first 4-8 weeks postpartum (Drackley, 1999). In order to ensure optimal health and performance during the subsequent lactation it’s imperative that dairy cows meet their nutritional and tissue metabolic requirements during this period (Grummer, 1995). Early lactation and negative energy balance (NEB) are associated with decreased progesterone, IGF-1, hypoinsulinemia and increased estradiol, GH, NEFA. Decreased insulin concentrations aid in the series of coordinated changes necessary to reduce glucose uptake by insulin responsive tissues (adipose and muscle) to facilitate glucose uptake within the mammary gland (Bauman and Elliot, 1983).
Cows suffering from severe NEB tend to have a more significant reduction in serum IGF-1 than their moderate NEB counterparts. This reduction is a direct consequence of the reduction in hepatic IGF-1 mRNA expression (Fenwick et al., 2008). This study also concluded that severe NEB cows exhibited reduced hepatic gene expression for the transcripts which encode IGF-1R, IGF-2R, and IGF binding proteins (IGFBP’s). Dairy cows experience a 70% reduction in IGF-1 following calving (Kobayashi et al., 1999) and is associated with increased plasma GH, decreased plasma insulin and the onset of NEB (Kim et al., 2004). GHR 1A and IGF-1 mRNA declines from 2 weeks before parturition to 1 week after parturition (Radcliff et al., 2003). The reduction in IGF-1 removes the negative feedback mechanism on GH synthesis and secretion allowing GH concentrations to increase postpartum (Veldhuis et al., 2001).
HOUSEKEEPING GENES AND HEPATIC GENE EXPRESSION
The use of housekeeping genes (HKG) such as glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) and cyclophilin as an internal standard when studying hepatic gene expression changes has been explored. Due to sensitivity the ideal HKG copy number will be stably expressed internal control gene and wont vary with experimental treatment or physiological status within the tissue of interest allowing increased accuracy in detecting changes in genes of interest (Thellin et al., 1999; Janovick-Guretzky et al., 2007). GAPDH catalyses the oxidative phosphorylation of glyceraldehyde-3-phosphate to 1, 3 bisphosphate during glycolysis and the reverse reaction in gluconeogenesis (GY, 2002). Cyclophilin plays a variety of different roles including molecular chaperones for protein folding (Ivery, 2000). Gene expression of GADPH and cyclophilin increases to 2-3 fold in early lactation while hyperinsulinemia reduces their expression (Rhoads et al., 2003). These genes are regulated within the liver so are not suitable for hepatic gene expressions studies during the transition period, negative energy balance or during times of plasma insulin variations (Rhoads et al., 2003). Recently RSP9 was found to be a suitable HKG for studies involving hepatic gene expression during the periparturient period as it was stably expressed across different sample types. However, concerns have been expressed over using a single HKG (Tricarico et al., 2002). For experiments where samples are taken pre-partum, postpartum or both and for experiments involving dietary treatments found that RSP9 and GAPDH would be the best combination of HKG for expression studies (Janovick-Guretzky et al., 2007).

GLUCOSE METABOLISM
Nearing the onset of parturition DMI declines (Greenfield et al., 2000b), in order to compensate for this decline the liver adapts through an increase in gluconeogenesis capacity from lactate, pyruvate, AA and other sources (Greenfield et al., 2000a). Phosphoenolpyruvate carboxykinase (PEPCK) and pyruvate carboxylase (PC) are rate limiting enzymes in hepatic gluconeogenesis for precursors entering the gluconeogenic path prior to triose phosphate. Through the tricarboxylic (TCA) cycle oxaloacetate is formed through the metabolism of propionate while the formation of oxaloacetate from lactate requires the conversion to pyruvate first by PC. Carbons present in the amino acids (AA) serine, cysteine, glycine, alanine, tryptophan and threonine are metabolised to glucose through the pyruvate step of the TCA cycle. However threonine can also be metabolized to glucose through propionyl-coA (Stryer, 1995). There is an increase in in PC mRNA during the transition to lactation which is reflected in alterations in PC activity and pretranslational regulation of PC mRNA abundance with no observed changes in PECK abundance until 28 days in milk (Weber et al., 2013). These alterations in glucose metabolism during the transition period favour an increased capacity for gluconeogenesis from lactate, amino acids or the two combined in order to facilitate the onset of lactation and the increased demand for glucose or gluconeogenic precursors (Greenfield et al., 2000a). All genes related to gluconeogenesis (Table 1) have been observed to have lower mRNA at -3 weeks relative to parturition compared with 13 weeks postpartum (Graber et al., 2010).
LIPID METABOLISM
Changes in key hormone concentrations involved in the somatotropic axis mentioned previously lead to increased fat mobilization increasing plasma NEFA and the rate of fatty acid uptake by the liver (Grummer, 1995). If the rate of hepatic liver triglycerides (TG) synthesis exceeds the clearance rate accumulation of TG occurs increasing the risk of fatty liver occurring. Clearance of TG occurs through hydrolysis and very low density lipoproteins (VLDL) synthesis. VLDL synthesis requires TG, phospholipids, cholesterol, cholesterol esters, apolipoproteins (B100, C and E) and microsomal triglyceride transfer protein (MTP) (Mensenkamp et al., 2001; Shelness and Sellers, 2001). Increase of MTP mRNA abundance is seen after calving but is not involved in the etiology of fatty liver. However, a significant decrease in ApoB100 is seen after calving and this may be consistent with the decreased synthesis and secretion of VLDL from the liver in periparturient cows (Bernabucci et al., 2004). The clearance of NEFA’s can be done by microsomal esterification (Zammit, 1999) through the activity of the mitochondrial form of glycerol phosphate acyltransferase (mGPAT). Hepatic mGPAT mRNA expression is decreased during the dry period and recovers 14 days after calving (Loor et al., 2005). ?-oxidation of NEFA in the mitochondria is controlled by the enzyme carnitine palmitoyltransferase (CPT-1). Activity and expression of CPT-1 increases between 1 and 14 days post-calving relative to 3 weeks prior to parturition (Dann and Drackley, 2005). The enzymatic capacity of NEFA ?-oxidation is thought to be under transcriptional control by the nuclear receptor peroxisome proliferator activated receptor-? (PPAR?). PPAR? controls mRNA abundance of several genes including CPT1A which increased sharply the day prior to calving (Loor et al., 2005). The same study found that mRNA abundance of ACOX1 which is encodes acyl-coA (flux generating enzyme of peroxisomal ?-oxidation of fatty acids) follows the same pattern as PPAR? after parturition with a gradual increase observed. An increase in the relative abundance in the genes involved in fatty acid metabolism between -3 and +13 weeks relative to parturition may play a role in the increased oxidation of fatty acids as a result of elevated NEFA(Emery et al., 1992). A similar pattern with the genes involved in gluconeogenesis was found genes involved in fatty acid metabolism (Table 1) at -3 and +13 weeks relative to parturition (Graber et al., 2010).
CHOLESTEROL SYNTHESIS
Genes including 3-hydroxy-3-methylglutaryl CoA reductase (HMGCR), = farnesyl diphosphate synthase (FDPS) and melavonate kinase (MVK) and low density lipoprotein receptor (LDLR) which are involved in cholesterol synthesis and uptake, liver X receptor (LXR-?) and its target genes involved in bile acid synthesis (CYP7A1) and cholesterol efflux in the liver were upregulated from 3 weeks pre-partum to 1 week postpartum (Schlegel et al., 2012). The synthesis and uptake of cholesterol within the liver is controlled by sterol regulatory element-binding protein 2 (SREBP-2); a member of the SREBP family; transcription factors involved in lipid synthesis and homeostasis (Brown and Goldstein, 1997). While the SREBP-2 expression levels weren’t affected in the aforementioned study, its target genes involved in cholesterol synthesis were upregulated during the transition from late pregnancy (-3 weeks) to early lactation (1 week) (Schlegel et al., 2012). The transcription of HMGCR controls cholesterol production as this is the rate limiting enzyme involved in its synthesis (Espenshade and Hughes, 2007). This enzyme was upregulated in the study carried out by Schlegel et al., 2012 proposes that at week 1 postpartum cholesterol synthesis could be stimulated. These studies indicate that cholesterol synthesis begins 3 weeks pre-partum and continues to increase till 1 week postpartum and is controlled at the level of transcription by the genes mentioned above and in Table 1.
EFFECTS OF INSULIN ON GENE EXPRESSION
Rapid decrease in insulin concentrations in periparturient cows is associated with a decrease in hepatic GHR 1A expression (Kobayashi et al., 1999). This was further shown when elevations in plasma insulin during NEB were shown to increase plasma IGF-1 which was associated with an increase hepatic GHR 1A and IGF-1 mRNA demonstrating insulin plays a key role in hepatic GHR expression and IGF-1 synthesis (Butler et al., 2003; Rhoads et al., 2004). Changes in GHR 1A expression play a critical role in nutrient partitioning. The increase in blood GH concentrations following parturition promotes lipolysis releasing NEFA which can be oxidized in the liver (Etherton and Bauman, 1998) suggesting the loss of GHR 1A may be key in ensuring lipid metabolism during lactogenesis (Radcliff et al., 2003).
Progesterone catabolism occurs primarily in the liver through the enzymes cytochrome P450 2C (CYP2C) and cytochrome P450 3A (CYP3A). Fractional rate progesterone decay within the liver can be reduced by insulin through its effects on CYP2C and CYP3A activity. The use of gluconeogenic feedstuffs or treatments involving insulin causes an 88% and 45% decrease in abundance of CYP2C and CYP3A mRNA respectively suggesting insulin plays a role in down regulating the mRNA for the cytochrome P450 family for progesterone catabolism (Lemley et al., 2008).
INFLUENCE OF BREED AND GENOTYPE ON HEPATIC GENE EXPRESSION
Variations in genetic selection principles within the dairy industry have led to a divergence of the Holstein Friesian breed. A comparison study between New Zealand Holstein Friesian (NZHF) cows and North American Holstein Friesian (NAHF) found that NZHF had greater abundance of IGF-1, and GHR 1A mRNA than their NAHF counterparts, this suggest the strain of Holstein Friesian can influence hepatic gene expression (McCarthy et al., 2009; Grala et al., 2011). Interestingly the study also found that suppressor of cytokine signalling (SOCS-3) a negative feedback molecule was greater in the NZHF strain. This coupled with the greater IGF-1 suggests enhanced coupling of the Jak/Stat pathway in the NZHF. There was however no effect of strain of Holstein Friesian on mRNA abundance of GHR 1A, IGF-2, IGF-R, IGF-2R and IGFBP 1 AND 6. This may be caused by greater uncoupling of the somatotropic axis in early lactation by the NAHF (Lucy et al., 2009).
The Fert (+), Fert (-) model developed by Teagasc examining cows with a high genetic merit for milk production but divergent in their genetic merit for fertility (Fert + = high genetic merit, Fert – = low genetic merit) found that Fert + cows fertility had increased expression of IGF-1 mRNA relative to the Fert – cows but this effect wasn’t seen until mid-lactation. Genotype had no effect on GHR1A, STAT5B, JAK2 or SOCS-3 (Cummins et al., 2012). The same study found that Fert- cows had greater expression of total GHR mRNA during the dry period and early lactation and this effect was not observed in mid to late lactation.
These studies demonstrate that breed strain and genotype can play a role in hepatic gene expression.
CONCLUSION
Continued research is necessary to determine the critical changes occurring in the transition period and the impact that genetic selection has in this critical time period. Increased understanding of the physiological and genetic changes in the liver could aid in reducing the disease complications and health problems associated with the transition period thus increasing the profitability of dairy cows.

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Table 1: Genes involved in the major functional roles of the liver

Gene Genebank ID Chromosome Gene type7 Expression Site8 Reference
Gluconeogenesis related gene1
PC 338471 29 PC LIV (Stryer, 1995; Weber et al., 2013)
PEPCK 282855 13 PC LIV (Stryer, 1995; Weber et al.,2013)
G6PC 538710 19 PC LIV (Graber et al., 2010; Weber et al., 2013)
PCCA 614302 12 PC LIV (Graber et al., 2010; Weber et al., 2013)
PCCB 515902 1 PC LIV (Graber et al., 2010; Weber et al., 2013)
FA Oxidation related gene2
ACSL 24494 3 PC LIV (van Dorland et al., 2009; Weber et al., 2013)
CPT 2 504502 3 PC LIV (van Dorland et al., 2009; Weber et al., 2013)
ACADVL 282130 19 PC LIV (van Dorland et al., 2009)
ACADM 505968 3 PC LIV (Graber et al., 2010; Weber et al., 2013)
Cholesterol synthesis genes3
HMGCR 407159 10 PC LIV (Graber et al., 2010; Weber et al., 2013)
MVK 505792 17 PC LIV (Schlegel et al., 2012)
FDPS 281156 3 PC LIV (Schlegel et al., 2012)
ACAT 1 511082, 15 PC LIV (Schlegel et al., 2012)
MTP 280868 6 PC LIV (Schlegel et al., 2012)
ApoB100 494004 11 PC LI V (Schlegel et al., 2012)
Fatty acid and triglyceride synthesis4
ATP citrate lyase 511135 19 PC LIV (Schlegel et al., 2012)
FASN 281152 19 PC LIV (Graber et al., 2010, Schlegel et al., 2012)
ACoC 281590 19 PC LIV (Graber et al., 2010)
GPAM 497202 26 PC LIV (Graber et al., 2010)
GPD2 504948 2 PC LIV (Graber et al., 2010)
Ketogenesis related gene5
HMGCS2 503684 3 PC LIV (Graber et al., 2010)
Nuclear receptor related gene6
PPAR? 281992 5 PC LIV (van Dorland et al., 2009)
SREBF1 539361 19 PC LIV (Graber et al., 2010)
1 PC = pyruvate carboxylase; PEPCK = phosphoenolpyruvate carboxykinase; PCK1 = cytosolic phosphoenolpyruvate carboxykinase; G6PC = glucose-6-phosphatase; PCCA = propionyl-coA carboxylase ?; PCCB = propionyl-coA carboxylase ?
2 ACSL = acyl-coA synthetase long chain; CPT 1A = carnitine palmitoyltransferase 1A; CPT 2 = carnitine palmitoyltransferase 2; ACADVL = acyl co-enzyme A dehydrogenase; ACADM = acyl-coA dehydrogenase medium chain;
3 HMGCR = 3-hydroxy-3-methylglutaryl CoA reductase; MVK = melavonate kinase;
4 FASN = fatty acid synthase; ACoC = acetyl-CoA-carboxylase; GPAM = glycerol-3-phosphate acyltransferase; GPD2 = glycerol-3-phosphate dehydrogenase
5 HMGCS2 = 3-hydroxy-3-methylglutaryl-co-enzyme A synthase 2; FDPS = farnesyl diphosphate synthase; ACAT 1 = acyl-CoA: cholesterol acyltransferase; MTP = microsomal triglyceride transfer protein; ApoB100 = apolipoprotein B100
6 PPAR? = peroxisome proliferators-activated receptor ?, SREBF1 = sterol regulatory element binding factor 1
7 LIV = liver; SI = small intestine;
8 PC = protein coding;