Physiol. Res. 70: 475-480, 2021

https://doi.org/10.33549/physiolres.934620

SHORT COMMUNICATION

Effects of H3 and H4 Histones Acetylation and Bindings of
CREB Binding Protein and p300 at the Promoter on Hepatic
Expression of γ-glutamyltransferase Gene in a Streptozotocin-Induced
Moderate Hypoinsulinemic Rat Model
Tomoyuki TANAKA1, Takahiro MIZUNO2, Tomoyuki NAKAGAWA1,2, Takashi
HAYAKAWA1,2, Masaya SHIMADA1,2
1

Division of Life Science for Food, Department of Life Science and Chemistry, Graduate School of
Natural Science and Technology, Gifu University, Gifu, Japan, 2Department of Applied Life
Science, Faculty of Applied Biological Sciences, Gifu University, Gifu, Japan
Received November 11, 2020
Accepted March 23, 2021
Epub Ahead of Print May 12, 2021

SummaryGamma-glutamyltransferase (GGT), a marker of liver disease, hasbeen shown to be associated with increased risk of diabetes andrelative insulin secretion deficiency. However, the mechanism ofhepatic Ggt regulation has not been explored fully. In this study,
we made a concerted effort to understand the mechanism byinvestigating the effects of acetylation of histones H3 and H4,
and bindings of histone acetyltransferases, CREB binding protein(CBP) and p300, at the Ggt promoter on the regulation of theexpressionofGgt gene in the livers of streptozotocin(STZ)-induced moderate hypoinsulinemia rat model. The rats
treated with STZ showed remarkably higher serum GGT level andhepatic Ggt/GGT expression than the untreated control rats.
Furthermore, the acetylation of histones H3 and H4, and thebinding of CBP not p300 at the Ggt promoter regions weresignificantly higher in the livers of STZ rats than those of thecontrol rats. These results suggest that an enhanced hepatic
expression of Ggt is associated with increased acetylation ofhistones H3 and H4 and CBP binding at the Ggt promoter in
STZ-induced moderate hypoinsulinemic rats.
Key words
Acetylated histone • CBP • GGT • Liver • Streptozotocin
Corresponding author
M. Shimada, Department of Applied Life Science, Faculty ofApplied Biological Sciences, Gifu University, 1-1 Yanagido,
Gifu-shi, Gifu 501-1193, Japan. E-mail: mshimada@gifu-u.ac.jp

Gamma-glutamyltransferase (GGT) plays a keyrole in the extracellular catabolism of the major
antioxidant, glutathione. Its increased circulating level byoxidative stress has been reported as a marker for hepatic
injuries, such as liver dysfunction induced by excessive
alcohol intake (Kunutsor 2016). Furthermore, humanobservational studies via a systematic review havereported that the circulating levels of GGT are associatedwith an increased risk of type 2 diabetes (Kunutsor et al.
2014). It has also been shown that the increased levels of
GGT in the blood are related to the attenuated function of
pancreatic β-cells in young obese subjects (Wang et al.
2013). However, in adult patients with type 1 diabetes,
Arkkila et al. (2001) could not establish its association
with diabetes duration, body mass index, and hemoglobin
A1c instead showed its association with retinopathy andneuropathy. Collectively, these studies suggest that theelevated circulating levels of GGT could be associatedwith relative but not absolute insulin secretion deficiency.
Furthermore, an in vitro study has shown that insulin
treatment decreases the expression of the GGT gene inhuman hepatocytes, HepG2 (Honma et al. 2017),
suggesting that increased insulin secretion downregulates
the expression of the GGT gene in the liver. However, theregulation of the expression of GGT is currently poorly
understood. Therefore, we speculate that a better
understanding of the regulatory mechanisms of hepatic

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476 Tanaka et al.
GGT expression under relatively deficient insulin
secretion could help develop the key strategies for the
management of diabetes.
Gene expression is regulated by histone
modifications, such as acetylation and methylation, andtranscription factors (Schübeler et al. 2004). In particular,enhanced gene expression is closely related to thehyperacetylation of histones H3 and H4 in theeuchromatin region of the genome (Roh et al. 2005).
In vivo studies using type 2 diabetic mice (db/db) models
have shown an increase in the hepatic expression of
gluconeogenic genes, such as phosphoenolpyruvatecarboxykinase 1 (Pck1), and acetylation of histone H3 on
the gluconeogenic genes (Ravnskjaer et al. 2013). In
addition, Suzuki et al. (2015) have reported that hepatic
expression of fatty acid synthase gene and acetylation ofhistones H3 and H4 on the fatty acid synthase gene are
increased in SHR/NDmc-cp rats, a spontaneously
hypertensive, obese and diabetic model of rats. However,
the effects of histone acetylation on the expression of the
Ggt gene in the liver of hypoinsulinemic models have notyet been reported.
In the present study, we examined the effects ofacetylation of histones H3 and H4 on the Ggt promoter instreptozotocin (STZ)-induced moderate (not severe)
hypoinsulinemia rat model on the expression of the
Ggt gene. Furthermore, it is known that CREB binding
protein (CBP) and p300, the histone acetyltransferasesbind to acetylated histones and nuclear transcription
factors to mediate the recruitment of the transcriptional
complex (Chan and La Thangue 2001). Therefore, toelucidate their role in regulating the expression of the
GGT gene, we investigated whether the bindings of CBPand p300 to the Ggt promoter are altered in the liver ofthe hypoinsulinemic rats.
Six-week-old male Wistar/ST rats werepurchased from Japan SLC (Shizuoka, Japan) and divided
into control and STZ groups. Control group rats (n=6)
were intraperitoneally administered a single dose of
saline. To induce moderate hypoinsulinemia, STZ grouprats (n=7) were intraperitoneally administered a single
low dose of STZ (40 mg/kg body weight; FUJIFILM
Wako Pure Chemical Corporation, Osaka, Japan)
dissolved in saline, prepared immediately (within 5 min)before the administration. At 5 days after treatment, we
confirmed that the random serum glucose level in the
STZ group rats was within the range of 300-500 mg/dl.
The rats were given free access to a laboratory chow diet(MF; Oriental Yeast, Tokyo, Japan) and water throughout
the acclimation and experimental periods. At 30 days

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after treatment, the rats were fasted for 4 h andeuthanized via cardiac puncture under isoflurane
inhalation anesthesia, and blood and liver tissue werecollected for subsequent assays. All animal care andexperimental procedures were approved by the Gifu
University Animal Care and Usage Committee.
Concentrations of serum biochemical parameters(glucose, triglyceride, insulin, glucagon, glutamic pyruvic
transaminase (GPT), and GGT) were measured using
commercial kits (Glucose CII Test Wako kit, TriglycerideE-test Wako kit, LBIS Insulin-Rat ELISA kit (U-E type),
Transaminase CII Test kit; all from FUJIFILM Wako
Pure Chemical Corporation, Glucagon enzyme
immunoassay (EIA) Kit; Yanaihara Institute, Shizuoka,
Japan, and GGT Activity Colorimetric Assay Kit; Bio
Vision, CA, USA). Hepatic triglycerides were determinedfollowing the method described by Shimada et al. (2019).
Hepatic total RNA was extracted and converted
to cDNA using commercial kits. Real-time PCR was
conducted as described by Shimada et al. (2019). The
primers used were as follows:
Pck1(5′-GATGACATTGCCTGGATGAA-3′,
5′-AACCGTTTTCTGGGTTGATG-3′),
Gpt(5′-CAGGAGGGCACCTATCATTT-3′,
5′-TTGGCATGGAAGTGACTGAG-3′),Ggt(5′-ACAGCCCAGATTGTGAAAGAC-3′,
5′-TCCGCACGATAGTTGTTAAGG-3′), and36b4(5′-CGAGAAGACCTCTTTCTTCCAA-3′,
5′-AGTCTTTATCAGCTGCACATCG-3′).
Relative mRNA levels were normalized to thehousekeeping gene 36b4 using the 2-ΔΔCT method.
Preparation of hepatic protein lysates andsubsequent immunoblotting were conducted following
Ichigo et al. (2019). The primary antibodies used wereanti-GGT antibody (Gene Tex, CA, USA) and antiTATA-binding protein (TBP; GeneTex). TBP was used
as a loading control.
Hepatic chromatin and subsequent chromatin
immunoprecipitation (ChIP) were prepared following themethod described by Shimada et al. (2019). The
antibodies used were as follows: anti-acetyl-histone H3(Millipore, CA, USA), anti-acetyl-histone H4 (Millipore),
CBP (Santa Cruz Biotechnology, TX, USA), p300 (Santa
Cruz Biotechnology), or normal rabbit/mouse IgG(FUJIFILM Wako Pure Chemical Corporation).
Immunoprecipitated DNA and input DNA were subjected
to real-time PCR. The primers, which amplified three Ggt
promoter regions, were as follows:
Ggt-900 (5′-CCTTTGAAGGGTTTTCCAGTG-3′,
5′-TCCTGGTGATGTCCACAGTTT-3′),

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Ggt-700(5′-CTTGTTGACCTTGGGCATCT-3′,
5′-GGACAGTTCCTTTGCCTCTTT-3′), andGgt-350 (5′-TGGAGATTCCAGACAGCATAGA-3′,
5′-TCACACAGATCTGAAGCCACTT-3′).
ChIP signals were normalized to thecorresponding input signals using the 2-ΔΔCT method.
Values are expressed as mean ± SEM. Differences between the two groups were evaluated using theStudent’s t-test. P<0.05 indicated statistical significance.
The body weight gain, levels of hepatic

Hepatic Regulation of Ggt in Moderate Hypoinsulinemia

477

triglycerides and serum insulin were significantly lower,
whereas the levels of serum glucose, triglycerides,
glucagon, GPT – another marker for hepatic damage, and
GGT were significantly higher in STZ rats than in control
rats (Table 1).
Hepatic expression levels of Pck1, Gpt, andGgt genes were significantly higher in STZ rats than in
the control rats (Fig. 1A). Similarly, the hepatic
expression level of GGT was significantly higher in
STZ rats than in the control rats (Fig. 1B).

Table 1. Physiological and biochemical parameters in control and STZ-induced moderate hypoinsulinemic rats.

Body weight gain (g)LiverWeight (g)Triglycerides (mg/g liver)
Serum
Glucose (mg/dl)
Triglycerides (mg/dl)
Insulin (pg/ml)
Glucagon (pg/ml)
GPT (U/l)
GGT (U/l)Control

STZ

132 ± 682 ± 7***

12.6 ± 0.4
53.6 ± 4.3

12.1 ± 0.4
35.9 ± 2.1**

141 ± 648.7 ± 3.6
564 ± 154369 ± 35
11.1 ± 0.5
1.25 ± 0.02

349 ± 31***74.8 ± 10.0*
66 ± 26**552 ± 41**18.1 ± 1.5**
1.86 ± 0.13**

Values are expressed as means ± standard error of the mean (n=6-7). *** P<0.001; ** P<0.01; * P<0.05, significantly different fromthe control group (Student’s t-test). Control, control rats treated with vehicle; STZ, moderate hypoinsulinemic rats treated withstreptozotocin; GPT, glutamic pyruvic transaminase; GGT, γ-glutamyltransferase.

Moreover, hepatic acetylation of histone H3 on
the -900 bp promoter region, acetylation of histone H4 on
the -900 bp, -700 bp, and -350 bp promoter regions, andbinding of CBP to the -900 bp and -700 bp promoter
regions in the Ggt gene were significantly higher in the
STZ rats than the control rats (Fig. 1C, 1D, and 1E),
whereas binding of p300 to the promoter regions did not
differ significantly between the two groups (Fig. 1F).
In this study, we observed higher hepatic
expression of Ggt/GGT and serum level of GGT in the
STZ rats than in control rats, which suggested that though
GGT is expressed in many tissues, the increased
GGT level in the blood could be partially attributable toenhanced hepatic expression of Ggt/GGT.
For the first time, this study revealed that thebinding of CBP and the acetylation of histones H3 andH4 were increased in the promoter regions of Ggt in theliver of the STZ-induced moderate hypoinsulinemic rats;
however, it remains unclear which signals contribute to

the enhanced hepatic expression of the Ggt gene. Shoukry(1988) showed that the treatment with insulin reduces the
GGT levels in the blood of STZ-induced type 1 diabetic
rats. In addition, Honma et al. (2017) reported that insulin
treatment decreases the expression of Ggt and Gpt genes
and gluconeogenic enzyme genes, such as Pck1, andacetylation of histones H3 and H4 on the Gpt andPck1 genes in HepG2 cells. Moreover, He et al. (2009)
demonstrated
that
insulintreatment
represses
Pck1 expression via phosphorylation of CBP at serine
436, which inactivates CBP in mouse liver. Collating thefindings of this study with those of previously reported
studies, it can be inferred that moderate hypoinsulinemia
could be associated with dephosphorylation of CBP atserine 436 and recruitment of CBP to increase acetylationof histones H3 and H4 in the promoter region of Ggt.
However, this should be validated through further studies
by investigating the effects of insulin administration to
STZ-induced moderate hypoinsulinemic rats.

478 Tanaka et al.

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Fig. 1. Hepatic expression of Ggt/GGT, acetylation of histones H3 and H4, and bindings of CBP and p300 to the Ggt gene promoter incontrol and STZ-induced moderate hypoinsulinemic rats. (A) Expression levels of Pck1, Gpt, and Ggt genes were analyzed using realtime quantitative PCR. Expression levels were normalized to the expression of 36b4. (B) Expression level of GGT was detected using
Immunoblot. The expression level was normalized to the expression of TBP. ChIP assays were performed using (C) anti-acetylated
histone H3, (D) anti-acetylated histone H4, (E) anti-CBP, (F) anti-p300, or normal IgG. ChIP signals were detected using real-time qPCR
with primers for the designated promoter regions of Ggt. Values are expressed as means ± standard error of the mean (n=6-7).
*** P<0.001; ** P<0.01; * P<0.05, significantly different from the control group (Student’s t-test). Control, control rats treated withvehicle; STZ, moderate hypoinsulinemic rats treated with streptozotocin.

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Furthermore, it is noteworthy that the moderate
hypoinsulinemic rats exhibited higher serum glucagon
levels in this study. It has been described that glucagon
increases the recruitment of CBP to hepatic
gluconeogenic genes under fasting conditions via
activation of the cAMP/PKA/CREB signaling pathway(Altarejos and Montminy 2011). Considering thesefindings, it is speculated that a combination of lower
insulin and higher glucagon could induce expression of
the Ggt gene via enhanced recruitment of CBP to theGgt gene; however, this hypothesis warrants further
investigation.
Though the present study demonstrated theenhanced expression of hepatic Ggt/GGT in STZ-induced
moderate hypoinsulinemic rats, the relationship between
moderatehypoinsulinemia
and
GGT
remains
controversial. Studies in humans have reported thatelevated GGT levels could predict the development of
insulin resistance (Lee et al. 2013, Ryoo et al. 2014).

Hepatic Regulation of Ggt in Moderate Hypoinsulinemia

479

Thus, further studies should chronologically investigate
circulating levels of GGT accompanied by pancreatic
β-cell exhaustion using young Goto-Kakizaki type 2
diabetic rats, which exhibit mild hyperglycemia witha reduction of β-cell mass at an early stage (Movassat
et al. 1995).
In conclusion, we have demonstrated thatenhanced hepatic Ggt expression is associated withincreased CBP binding and histone H3 andH4 acetylation on the Ggt gene promoter regions in
STZ-induced moderate hypoinsulinemic rats.

Conflict of Interest

There is no conflict of interest.

Acknowledgements

This work was supported by JSPS KAKENHI Grant
number JP18K02237.

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