Gene 574 (2015) 48–52

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Gene

journal homepage: www.elsevier.com/locate/gene

Research paper

A SNP in the 3′-untranslated region of AMPKγ1 may associate with

serum ketone body and milk production of Holstein dairy cows
Ahmad Mahmoudi a,1, Amir Zargaran a,1, Hamid-Reza Amini a,1, Assad Assadi b,
Reza Vajdi Hokmabad b, Shahin Eghbalsaied a,⁎
a
Young Researchers and Elite Club, Isfahan (Khorasgan) Branch, Islamic Azad University, Isfahan, Iran
b
Department of Animal Science, Miyaneh Branch, Islamic Azad University, Miyaneh, Iran

a r t i c l e i n f o a b s t r a c t

Article history: AMPK is the key switch for providing the energy balance between cellular anabolic and catabolic processes.
Received 9 July 2015 In this study, we aimed to screen the PRKAG1 (AMPKγ1) gene in high, moderate, and low producing Holstein
Accepted 23 July 2015 dairy cows. A sample of 100 pregnant dairy cows, comprising 41 high, 33 moderate, and 26 low milk yields
Available online 27 July 2015
were selected from three large dairy herds in Isfahan province of Iran. Body condition score (BCS) was estimated
before parturition while beta hydroxyl butyric acid (BHBA) as a measure of ketone bodies was measured at the
Keywords:
AMPK
fifth day postpartum. In addition, using three primer pairs covering exons 2–11 and 3′-UTR of the PRKAG1 gene,
BHBA a random sample of 10 high milk yield dairy cows were amplified and sequenced. The sequencing results showed
mir-423-5p the presence of a T12571C mutation in intron 6 and a T14280C mutation in the 3′-untranslated region (UTR) of
MicroRNA the PRKAG1 gene. Following a PCR reaction for amplification of the 3′-UTR amplicons, single strand conformation
PRKAG polymorphism (SSCP) assay was implemented for discrimination of the mutation in the studied population.
Ketosis Then, we evaluated if the mutation associates with the BCS, serum BHBA level, and production traits. The
experimental analysis showed that the mutated allele significantly increased the BHBA level, BCS, as well as
milk and protein yield. Bioinformatic study revealed that this 3′-UTR mutation distorts the target site of
mir-423-5p microRNA which is one of the most highly expressed microRNAs in the bovine mammary gland,
liver, and kidney. Given the role of AMPK in energy metabolism, the newly identified 3′-UTR mutation highlights
the importance of AMPK and suggests a role of miRNAs for regulation of cellular metabolism, metabolism
disorders, and production traits in Holstein dairy cows.
© 2015 Elsevier B.V. All rights reserved.

1. Introduction circadian oscillation, and physiological and subsequently seasonal be-

The transition period, three weeks pre- and post-calving, is the most
decisive phase for the health and milk production of dairy cows
(Drackley, 1999). Since energy requirements increase dramatically
during this period, considering the ability of the dairy cow to cope
with the metabolic needs is a very important factor for improving
managerial issues of dairy cows, particularly some metabolic disorders
such as ketosis and fatty liver syndromes (Block, 2010).
Adenosine mono-phosphate protein kinase gene (AMPK) was intro-
duced as a “fuel gauge” which switches anabolic and catabolic pathways
(Hardie and Carling, 1997) and is involved in cellular homeostasis,
Abbreviations: ABCI, animal breeding center of Iran; AMPK, adenosine mono
phosphate protein kinase; BHBA, beta hydroxyl butyric acid; BCS, body condition score;
CDS, coding DNA sequence; miRNA, microRNA; NCBI, National Center of Biotechnology
Information; RPM, read per million; SNP, single nucleotide polymorphism; SSCP, single
strand conformation polymorphism.
⁎ Corresponding author.
E-mail address: shahin.eghbal@khuisf.ac.ir (S. Eghbalsaied).
1
These authors contributed equally to this work.
havioral events and daily environmental changes (Vieira et al., 2008).
Therefore, the study of this gene family highlights AMPK's roles in the
adaptation process, production, reproduction, and longevity attributes
of animals as well as some human syndromes such as insulin resistance
and abdominal obesity.
Mammalian AMPK comprises of two catalytic subunits (α1, α2) and
five non-catalytic regulatory subunits (β1, β2, γ1, γ2, γ3) (Hardie and
Carling, 1997; McKay et al., 2003; Xiao et al., 2007). Each of the three
gamma components includes four cytathione beta-synthase domains
which bind the adenosine part of AMP (Hardie and Hawley, 2001).
Therefore, any mutation in the AMPKγ1 isoform leading to a small
modification in the adenyl-binding site can severely affect AMP and
ATP binding to AMPK (Xiao et al., 2007). AMPKγ1, PRKAG1 in bovine,
consists of 12 exons and 11 introns and transcribes a 1619 bp mRNA
which comprises of a coding DNA sequence (CDS: 993 nt) and a very
long 3′-UTR (627 nt) (Benkel et al., 2005).
Apart from modifications in the protein structure through mutations
in the coding sequence, administration of gene expression is the other
important mechanism for the regulation of a protein function. Most of

http://dx.doi.org/10.1016/j.gene.2015.07.077
0378-1119/© 2015 Elsevier B.V. All rights reserved.
 A. Mahmoudi et al. / Gene 574 (2015) 48–52
the mammalian genes are transcribed from both DNA strands and trans-
lated into proteins whereas the remaining transcripts are considered
non-coding RNAs (ncRNAs) (Carninci et al., 2005). These types of RNA
can act as activators or repressors for other genes (Qu and Adelson,
2012). Among ncRNAs, microRNAs, known as miRNAs, are a class of
small RNAs (19–25 nucleotides) with cap and polyA tail (Winter et al.,
2009) which interfere with gene expression via either mRNA degrada-
tion or translational repression (Galio et al., 2013). Over the past decade,
administration of miRNAs has been a nascent technology for the regula-
tion of gene expression. These elements can down regulate the transla-
tion step by triggering RNAse endonucleases and enhancing mRNA
decapping and de-adenylation (Valencia-Sanchez et al., 2006). So far,
around 1000 miRNA genes have been discovered in mammalian
genomes, though the function of the majority of these known miRNA
still remains to be discovered (Glazov et al., 2009). In silico evaluation
of bovine genome indicated a majority class of unknown miRNAs
which are specific to ruminants (Glazov et al., 2009; Qu and Adelson,
2012). Nearly 60% of bovine ncRNAs are intergenic and highly
corresponded to the 3′-UTRs of messenger RNAs (mRNAs) (Qu and
Adelson, 2012). Moreover, there are a high number of intergenic
ncRNAs which act through cis-regulation of mostly regulatory genes
and locate in the 1 kb proximity of UTRs (Qu and Adelson, 2012).
As the 3′-UTR associated/proximate ncRNAs are in highly conserved
regions (Qu and Adelson, 2012), targeting the 3′-UTR of animal mRNA
has been one of the most important pathways for regulation of gene
expression (Glazov et al., 2009).
In this study, we aimed to screen the bovine PRKAG1 exons 3–7 and
12 as well as the 3′-UTR and evaluate the effect of polymorphisms on
305-day production traits, serum BHBA level, and BCS of Holstein
dairy cows.
2. Materials and methods
2.1. Animals and records
In this study, based on the first parity adjusted milk yield records for
a 305-day production period, 41 high- (from 13,000 to 20,000 kg),
33 moderate- (from 11,500 to 12,900 kg), and 26 low- (from 7500
to 11,400 kg) producing dairy cows were selected among available
pregnant dairy cows from three large dairy herds in Isfahan province
of Iran. Since assessment of the ketosis prevalence was one of the
main agendas of this research, the selected dairy cows were restricted
to those having more than three parities. Evaluation of BCS was carried
out at the late stage of the pregnancy, during the dry period. At the fifth
day post-partum, at 7 am, blood samples were collected and quickly
sent to the laboratory for the measurement of serum BHBA level using
the D-3-hydroxybutyrate (RANBUT) assay, RB-1007 kit (Randox, UK).
A fraction of the collected blood underwent the DNA extraction process.
Moreover, first parity records of production traits such as milk, fat,
and protein yield which were previously adjusted for 305-days of
production period were gathered from Animal Breeding Center of Iran
(ABCI). Because the maximum heritability for production traits is
attributed to the first parity records, the genetic polymorphism effects
were evaluated on the first parity productive traits.
2.2. Genotyping assays
The polymerase chain reactions (PCR) assay was performed using
three primer pairs which were designed to amplify exons 3–7 which
are closely located with quite short introns, as well as exon 12, which
is the last exon of PRKAG1, and the early part of the 3′-UTR of PRKAG1
(Table 1) based on the AC_000162.1 reference sequence in National
Center for Biotechnology Information (NCBI). The first, second and
third primer pairs cover exons 3–5, exons 5–7, and exon 12 along
with the early part of 3′-UTR, respectively. PCR productions of the
amplified fragments from 10 randomly selected high-yield dairy cows
49
Table 1
Primer pairs for amplification of exons 3–7, 12 and 3′-untranslated region (UTR) of
bovine PRKAG1.
Primer sequence Amplicon length
F1: 5′-AATAGCAACAAAACAGAATAC-3′ 847 bp: from 11,654 to 12,500
R1: 5′-CAACAGAACTCGAGACTCA-3′
F2: 5′-TTATTGGCATCTTGTACTGGG-3′ 757 bp: from 12,245 to 13,001
R2: 5′-GGAAGGCAGGGATTAATGG-3′
F3: 5′-AGGGTGGGGACTGACGG-3′ 384 bp: from 14,053 to 14,436
R3: 5′-GATGGGCTGGGTGAGGATAAG-3′
were sequenced (BioBasic, Canada). The sequencing results were
aligned to the reference sequence and possible mutations were looked
for by the CLC Workbench 6 software. All of the DNA samples were
genotyped for the detected mutation using the single strands conforma-
tion polymorphism (SSCP) technique considering the sequenced
samples as positive controls. Since the detected mutation was in the
3′-UTR of PRKAG1, we used the MiRbase database (http://www.
mirbase.org/search.shtml) to search for miRNAs which target the
mutated sequence.
2.3. Statistical analysis
Data were analyzed using the General Linear Model (GLM)
procedure in SAS package. Mean comparison among different
genotypes for the afore-mentioned traits were carried out using
the LSD test. Allelic and genotypic frequencies were compared
using Chi-square test. The P-value b0.05 was considered as the
significant level for comparison-wise error rate.
3. Results
3.1. Detection of two mutations in the PRKAG1 of Holstein dairy cows
Alignment of the sequenced samples to the NC_007303.5 reference
number, Bos taurus breed Hereford chromosome 5 alternate assembly,
in NCBI showed the presence of two mutations (Fig. 1). The first single
nucleotide polymorphism (SNP) was T12571C which is located in
intron 6 of PRKAG1. The second SNP was T14280C which resides in
the 3′-UTR of PRKAG1; or T1043C compared to the NM_174586.2
mRNA sequence in NCBI.
Then, the genotype of all 100 dairy cows for this 3′-UTR polymor-
phism was detected via the SSCP technique. Allelic and genotypic
frequencies for the T1043C mutation are presented in Table 2. Allelic
frequency of the SNP was 45.5% in the whole selected population. The
genotypic results indicated higher frequencies for both wild- and
mutant-type homozygotes whereas the heterozygote genotype was
considerably lower than the expected frequencies based on the
Hardy–Weinberg equilibrium. The majority of high producing dairy
cows were mutant type homozygous for the 3′-UTR SNP. The genotypic
distribution was significantly different among high-, moderate-, and
low-yield classes.
3.2. The 3′-UTR mutation associates with variation in serum BHBA level,
BCS, and production traits
The mean body condition score of pregnant dairy cows at the late
dry period was significantly higher in CC genotypes versus both TC
and TT classes (Fig. 2). Then we tested if the serum BHBA level at the
5th day following the parturition was different among the PRKAG1 ge-
notypes (Fig. 2). A highly significant difference was observed between
the homozygous mutant type (0.77 mmol/L) compared to both hetero-
zygous and wild type homozygous genotypes (0.56 mmol/L). Moreover,
mean comparison among different genotypes for the 305-day produc-
tion traits are presented in Fig. 3. In agreement with the observed signif-
icant differences in the genotypic frequency distribution among high-,
50 A. Mahmoudi et al. / Gene 574 (2015) 48–52

Fig. 1. The presence of T12571C mutation in intron 6 (A) and T14280C in 3′-untranslated region (UTR) (B) of bovine PRKAG1 gene.

moderate-, and low-yield groups, 305-day milk yield was also signifi- exist in intron 6 and 3′-UTR, respectively. These mutations have not
cantly different among the three defined genotypes. The mutant type al- been reported by other groups. The abundance of T14280C mutation,
lele was associated with the higher 305-day milk and protein yield, corresponding to T1043C in the PRKAG1 mRNA, was evaluated in a pop-
although the difference was not significant between the mutant type ulation of 100 dairy cows with high, low and moderate milk production.
homozygous and the heterozygous classes for the protein yield trait. The mutated allele was considerably more prevalent in high milk yield
The numerically lower fat yield in the wild type homozygous class dairy cows whereas the wild type allele was highly distributed in the
was not statistically different with two the other genotype classes. moderate and low milk yield dairy cows. The results of a previous
study on bovine PRKAG1 gene indicated that there are four mutations
3.3. The T1043C SNP alters target mRNA for miRNAs in the intron 9 of this gene in Angus, Charolis, Herford, Simmental,
Limousin, Holstein, Waguya and Brahman cows as well as bisons
Searching for the 3′-UTR anti-sense base-pairing through the miRNA (Benkel et al., 2005). The analysis of the data from the current study in-
database on the miRBase website indicates that the bta-miR-423-5p dicated that there was a significant difference between the homozygous
sequence is complimentary to the 3′-UTR of mRNA at the wild type mutant animals and other genotypes for the serum BHBA level and BCS
status (Table 3). However, in case of the T1043C mutation, the 3′-UTR as well. It has been well-documented that AMPK is a very important
is no longer a target site for bta-miR-423-5p but is base-paired to the controlling factor for fatty acid synthesis and breakage. It has been sug-
bta-miR-2453 and bta-miR-2292. gested that the importance of PRKAG1 sub-unit is due to the nucleotide
binding sites which contain a permanently bound AMP molecule and
4. Discussion two highly competitive sites for ADP and Mg-ATP (Xiao et al., 2007).
There are several mechanisms by which AMPK can switch and manage
In this study, we used the PRKAG1 gene, a main component of the cellular metabolisms. Using knockout mice for the PRKAG gene, it
the AMPK factor which is the fuel gauge of cellular metabolism, as a has been evident that one of the AMPK action mechanisms is through
candidate gene for controlling energy production and consumption an intervention on the circadian clock mechanism (Vieira et al., 2008)
during the transition period of Holstein dairy cows. We evaluated BCS which is a key regulator for feed intake and cellular metabolism.
and BHBA production as important factors for dairy cows' metabolic It has also been verified that mutations in the AMPKγ3 gene are associ-
disorders such as ketosis and fatty liver syndrome. The analysis of the ated with glycogen content of skeletal muscles (Milan et al., 2000).
sequenced samples for the PRKAG1 gene showed that there were two Decrease in the hypothalamic levels of AMPK reduced feed intake and
SNPs segregating in the population; T12571C and T14280C which subsequently weight gain while increasing AMPK induced obesity
(Minokoshi et al., 2004). In addition, it has been shown that hypotha-
Table 2 lamic AMPK regulates feed intake and body weight through the uptake
Allelic and genotypic frequencies for T1043C SNP in the 3′-untranslated region (UTR) of and oxidation of fatty acids and glucose (see (Xue and Kahn, 2006) for
bovine PRKAG1.
review). In accordance with our results for BCS and BHBA, activation
Milk yield class Genotype frequencies (%) Allelic and inhibition of AMPK inhibited and enhanced the PPARγ expression,
frequencies respectively, and thereby directed adipogenesis in sheep (Tong et al.,
CC⁎ TC TT C T 2008). Administration of PPARα before parturition associated with
High 27 (65.8) a
9 (22.0)a
5 (12.2) a
76.8a
23.2c feed intake, fatty acid oxidation, and BHBA production (Smith et al.,
Moderate 6 (18.2)b 9 (27.3)a 18 (54.5)b 31.8b 68.2b 2007). There is a delicate relation between BCS before parturition and
Low 2 (7.7)b 3 (11.5)b 21 (80.8)b 13.5c 86.5a ketogenesis after parturition. AMPK can activate fatty acid synthesis
⁎ Means in each column with different letters are significantly different at P-value b0.05 by acetyl-CoA carboxylase and HMG-CoA reductase and in parallel
using Chi-square test. activates lipase and glycogen synthase which regulates lipolysis and
 A. Mahmoudi et al. / Gene 574 (2015) 48–52
Fig. 2. Mean comparison of BCS and serum beta-hydroxy butyric acid (BHBA) level among different genotypes for T1043C in 3′-untranslated region (UTR) of bovine PRKAG1. Means with
different letters are significantly different at P-value b0.05.
glycogen synthesis, respectively (Hardie and Carling, 1997), and finally
affect the BCS of the dairy cows. On the other hand, in circumstances of
excess malonyl-CoA, AMPK inhibits acetyl-CoA carboxylase and causes
fatty acid synthesis inhibition and fatty acid oxidation, and finally
increases ketogenesis (Hardie and Carling, 1997; Blázquez et al., 1999).
The current study also revealed a significantly higher milk yield for
dairy cows carrying the mutant alleles of the 3′-UTR in a quite additive
manner. This increase in milk production can be attributed to an in-
crease in the appetite and subsequently energy and protein production.
The protein yield was also higher in the homozygous mutant compared
to the other genotype classes. However, there was no significant differ-
ence in the fat yield of dairy cows carrying the mutant alleles compared
to the wild type one. In agreement with this result, AMPK activation in
caprine mammary epithelial cells did not increase fatty acid synthesis
(Zhang et al., 2011). Finally, we searched for a possible mechanism by
which the T1043C mutation in 3′-UTR of PRKAG1 mRNA can influence
the AMPK production/activation and the subsequent variation in the
serum BHBA, BCS, and production traits of dairy cows. Interestingly,
the sequence containing the wild type nucleotide seems to be a target
for mir-423-5p. However, the altered sequence incorporating the
mutant SNP was no longer a target site for mir-423-5p. Instead, this
new sequence was the target for two other miRNAs, namely mir-2453
and mir-2292.
We need to consider two main issues regarding the miRNAs. Firstly,
how well does the miRNA match the target mRNA? Matching of a
miRNA to a mRNA at a specific site can direct endonuclease cleavage
of the mRNA template by activation of slicers and argonaute proteins
(Valencia-Sanchez et al., 2006). The results of our study showed that
the mir-423-5p, mir-2292, and mir-2453 do not completely match at
their seed region with the target mRNA. In spite of the Watson–Crick
pairing to the miRNA seed, animals miRNAs are scarcely fully
Fig. 3. Mean comparison of 305-day production traits among different genotypes for T1043C in 3′-untranslated region (UTR) of bovine PRKAG1. Means with different letters are
significantly different at P-value b0.05.
51
complimentary to the target mRNA and subsequently are rarely
cleaved by endonucleases and the Ago2 protein system (Yekta
et al., 2004; Mullokandov et al., 2012). Instead, the non-silencing
system by translational inhibition and destabilization induction is the
main approach for the regulation of expression (Mullokandov et al.,
2012). For example, the let-7 and lin-28 miRNA families have imperfect
seed pairing (Lewis et al., 2005; Wu and Belasco, 2005). Apart from the
seed pairing, supplementary pairings to nucleotide 13–16 and 11–18
can compensate for single nucleotide mismatches at the seed region
nucleotides and direct argonaute-mediated cleavage (Yekta et al.,
2004; Jing et al., 2005; Bartel, 2009). The mir-423-5p has a 7-mer
pairing from 5–11 and two extra supplementary pairing at nucleotides
13–14 and 16–18 which can support efficient interaction of the miRNA
and the target mRNA. Moreover, the mir-2292 and mir-2453 associate
with a series of matching/mismatching at nucleotides 4–22 and 3–20,
respectively. Secondly, how much does the miRNA express in the target
tissue? Using a high throughput assay and a devising technology of
miRNA sensor decoy library, it has been found that the majority of sup-
pressed targets correspond to miRNA clusters with N1000 cumulative
RPM though miRNA with b 100 RPM are not capable of modulating
the target regulation (Mullokandov et al., 2012). Based on the current
literature, mir-2292 and mir-2453 are undetectable or detectable
at b6 RPM in the bovine fetal and adult cells, as well as the bovine mam-
mary gland (Sun et al., 2014), so they were classified as non-functional
miRNA. On the other side, mir-423-5p is highly conserved and
expressed in fetal and adult bovine cells (Sun et al., 2014). It was ranked
among the top 10 highly expressed miRNAs in the follicular fluid ob-
tained from bovine growing oocyte (Sohel et al., 2013), bovine kidney
(Glazov et al., 2009), and bovine liver as well (Fatima et al., 2014).
Moreover, this miRNA has been classified among the top 30 highly
expressed miRNAs with more than 15,000 RPM in bovine mammary
52 A. Mahmoudi et al. / Gene 574 (2015) 48–52
Table 3
miRNA base-pairing to the 3′-untranslated region (UTR) of bovine PRKAG1 using the
miRBase website: http://www.mirbase.org/search.shtml.
*The highlighted base is the single nucleotide polymorphism location.
glands (Le Guillou et al., 2014). Therefore, we suppose that in low and
moderate producing dairy cows, expression of PRKAG1 is partly
controlled by mir-423-5p as a highly expressed miRNA. However, the
mutated allele alters the mir-423-5p target sequence and dramatically
decreases the miRNA affinity to the PRKAG1 mRNA. On the other
hand, provision of new targets for two other miRNAs are less likely
as important as the wild type target base-pairing since the expression
of these miRNAs are very lower than the truncation level for
miRNA effectiveness.
5. Conclusion
This study revealed the presence of one SNP in the 3′-UTR of the
bovine PRKAG1 gene. The frequency of the mutated allele was remark-
ably more abundant in dairy cows with high milk yield compared to
the moderate and low milk yield groups. In addition, this mutation
caused to an increase in the BCS prior to parturition and serum BHBA
level. Further bioinformatic analysis revealed that this part of 3′-UTR
is a target site for mir-423-5p which is one of the most abundantly
expressed miRNA in liver, kidney, and mammary gland of dairy cows.
We suppose that PRKAG1 expression can be regulated through this
highly expressed miRNA and this affects AMPK production. However,
further experiments are needed to obtain experimental proof for this
suggested action mechanism.
Acknowledgments
Authors would like to appreciate Dr. Stefan Wagner for editing this
manuscript. This project was funded by the Isfahan (Khorasgan) Branch,
Islamic Azad University, Isfahan Iran (13920210).
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