VALPROIC ACID ADMINISTRATION IN PREGNANT RATS IN INHIBIT

INSULIN SYNTHESIS ON THEIR BABIES PANCREATIC BETA CELL

Valproic acid through the activity of histone deacetylase (HDAC), can affect

gene expression during pancreatic organogenesis, which affects the development of

pancreatic beta cells to produce insulin. The study using Sparague Dawley rats aged

3-4 months, were 30 females, body weight of 200-250g, divided into 4 groups. Group

1 was given distilled water as a control group. Treatment group 2-4 was given 250mg

of valproic acid on gestational age day 10, day 13 and day 16. A total of 84 babies

mice was observed for concentrations of DNA, RNA, and the RNA/DNA ratio,

insulin, and glucose, as well as immunohistochemical insulin and glucagon pancreatic

tissue with four weeks interval, starting from week 4 through week 32. The results

showed that the babies from valproic acid treatment rats decreased the concentration

of DNA, RNA, the RNA/DNA ratio, and insulin (p <0.05), supported by low

Immunoreactive to the insulin, resulting in elevated levels of glucose (p <0.05 ) in

babies from treatment group. Valproic acid administration in gestation age day 10, day

13, and day 16s induce the expression of genes that play a role in pancreatic

organogenesis, thus causing damage to the beta cells that producing insulin in the

baby mice, indicated by a decrease in secretion and insulin levels.

Introduction

Pancreatic beta cells are cells that play a role in the synthesis and secretion of

insulin (Fu et al., 2013), any defect in the beta cell development during organogenesis

process will affect the ability of beta cells to synthesize and secretion of insulin

(Aguirre et al., 2015). Insulin plays a role in maintaining the balance of blood glucose

levels (Gerace et al., 2015), a disturbance due to a decrease in the number of insulin

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secretion, insulin receptors malfunction or a combination of both (Pagliuca et al.,

2014) can lead to conditions of hyperglycemia, which is a feature of diabetes mellitus

(Inzucchi et al., 2012), especially type 2 diabetes, when the diagnosis is made, the

functional capacity of beta cells has fallen by up to 80% of the original capacity

(Effendi and Waspadji 2011).

Pancreatic development is regulated by specific control derived from the

differentiation of progenitor cells and is influenced by signaling and transcription

pathways (Aguirre et al. 2015). The development of the pancreas include two

transitional regulations that begins on day 8.75 to day 12.5 with the formation of buds

on the endoderm layer, the cells proliferate and increase in size quickly. In addition,

there is a change morphogenesis tubular structure called the primary transition. In this

transition, epithelial cells have not undergone a process of differentiation (Pan and

Wright 2011). Meanwhile, in the transition secondary regulation, pancreatic epithelial

expand the branching followed by the process of differentiation of endocrine cells,

acinar and duct that occurred on day 12.5 to birth (Shih and Sander 2013).

The use of valproic acid during pregnancy, can interfere the expression of

genes that play a role in the development of the pancreas such as the expression of the

Pdx1 gene which acts as a central onset of signaling complex and transcriptional

regulation tissue that regulate the proliferation and differentiation of pancreatic

(Mastracci and Molina 2014), Nkx6.1 gene expression on tip and tunk branching. Tip

part will develop into the exocrine pancreas, while tunk will develop into pancreatic

endocrine (Mastracci and Molina, 2014). Ngn3 gene expression that play a role in

expressing progenitor cells during the development of pancreatic beta cells (Sostrup et

al. 2014).

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 Exposure to valproic acid during pregnancy, especially during organogenesis

will affect embryo development (Ornoy and Ergaz 2010). Abnormalities that occur

can be abnormality of structure, function, or metabolic organ that seen after birth.

This occurs because of specific factors to the material that affects the proliferation and

differentiation of cells during embryonic development (Mamashli et al., 2011).

Valproic acid is an antiepileptic drug that affects histone modifications during the

process of cell proliferation and differentiation, by affects the activity of histone

deacetylase (HDAC) (Christian et al., 2012). Structural modifications of histones can

be done by acetylation or deacetylation that important in modulating gene expression

(Ververis et al. 2013), played by histone acetyl transferase (HAT) and HDAC. HAT

enzymes loosen the DNA conformation which causes the transcription (activation),

while HDAC tighten DNA conformation which causes inactivity transcription

(repression) (Prasetia 2011). The purpose of this study is to investigate the ability of

valproic acid in affecting the rats fetal organogenesis and the effects on the

development of pancreatic beta cells that play a role in the synthesis of insulin.

Method

Experiments Design and Data Analysis

The study design used completely randomized design (CRD) factorial 4 x 7,

which consists of two main factors, the four factors that consists of a group that was

not given valproic acid and a group that given 250 mg valproic acid in gestational age

day 10, day 13, and day 16, as well as seven factors in growth periods, 4, 8, 12, 16,

20, 24, and 32 weeks. Each treatment consists of three replicates. Data were analyzed

using analysis of variance (ANOVA). All of the data analysis done by the general

linear models procedure in SAS v9.4 program.

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RESULTS

Valproic acid (VPA) during organogenesis able to influence the process of

proliferation and differentiation of the pancreas cells, represented with low

concentrations of DNA from the parent pancreas of group 250 mg valproic acid.

Decreasing the concentration of mice DNA pancreas from treatment group at

gestational age day 10 of 7,43%, day 13 7,54%, and day 16 of 6,06%, compared with

controls (P <0.05), during the growth period, decreased DNA concentration of 5,18%

occurs, compared to the early age of observations (4 weeks). Decreasing the

concentration of DNA followed with a decrease in RNA concentration pancreas of

mice from group treatment parent at gestational age day 10 of 15,05%, day 13 of

11,09%, and day 16s of 10,21%, compared with controls (P <0.05). RNA pancreas

concentration decreased during a growth period occurred in the week 20 of 4.81%,

compared to the early age of observation (P <0.05). DNA and RNA pancreas

concentrations decreased in mice from the treatment group directly reduce the

RNA/DNA pancreas ratio of 3,85%, 8,46%, and 4,61% (P <0.05), respectively, but

did not indicate a significant change in the ratio during growth periods (Table 2).
a-c
different superscript in the same row showed a significantly different (P <0.05);
a-d a-
different superscript in the same column sowed a significantly different (P <0.05);
k
different superscript in rows and columns significant differences (P <0.05).

The ability of valproic acid in influencing gene expression during

organogenesis, resulted in decreased of RNA ability to synthesize protein, which is

observed with low concentration of pancreatic insulin of 54.24%, 45.41% and

40.95%, in babies mice from the valproic acid parent group at gestational age day 10,

day 13, and day 16s, compared with controls (P <0.01). A decrease in blood insulin

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levels, resulting in an increase in blood glucose levels significantly. Blood glucose

levels in mice from parent that given valproic acid at gestational age day 10, day 13,

and day 16 increased significantly of 26,83%, 28,28% and 22,81% compared with

controls (P <0.05). Blood glucose levels during growth periods reflects the increasing

previously followed by a decrease in blood glucose levels of mice in all groups.

Immunohistochemical staining observations in pancreatic tissue sections of

babies mice in all groups is done by observed the population of beta cells and alpha

cells, which were analyzed descriptively through appearance of immunoreactive

insulin and glucagon from the beta cells and alpha cells undergoing changes. The

babies mice beta cells from control group showed a high immunoreactive to insulin,

compared to the babies mice beta cells from the treatment group, that showed lower

immunoreactive to insulin. In babies mouse from day 13 treatment group (T2),

Immunoreactive to insulin is lower when compared to other treatment groups. While

the babies mice from the day 10 treatment group (T1) and day 16 treatment group

(T3) showed a lower immunoreactive to insulin than the babies mice from control

group, but showe a higher immunoreactive to insulin than the babies mice from day

13 treatment group (T2) (Figure 2).

DISCUSSION

Valproic acid is an anticonvulsant drug with a broad spectrum in the treatment

of epilepsy (Khan et al., 2011). The use of VPA during pregnancy is known to cause

congenital malformations (Indrawati 2012), this was due to the 80%-94% VPA is able

to bind the plasma proteins (Guerrini 2006), which can be transferred through the

placenta in both animals and humans (Kumar et al. 2000). Susceptibility to

abnormalities depends on the period of development of different organs during

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pregnancy (Ekwere et al., 2011). Beside the dosage, route of drugs administration and

drug agency itself (Polifka and Friedman 2002), it was concluded that the embryo

damage is formed by multifactorial contribute to both environmental and genetic

factors (Wlodarczyk et al., 2011).

Valproic acid administration during gestational age day 10, day 13 and day 16,

with a dose of 250 mg, was associated with the genes expression that play a role in the

development of the pancreas, particularly of pancreatic beta cells. Valproic acid

administrations at day 10s gestation associated with expression of Pdx1 gene that

play important role in the early steps of pancreatic development (Otsuka et al. 2015).

Day 13 pregnancy, associated with the expression of Nkx6.1 gene, which plays a role

in beta cell neogenesis through transdifferentiation of pancreatic progenitor cells, this

process determines the number of beta cells at birth (Schwartz et al. 2010). Loss of

Nkx6.1 gene expression result in decreased the number of pancreatic beta cells. On

day 16, the gestation associated with Ngn3 gene expression, which is expressed on the

formation of the islets of Langerhans (Gomez et al. 2015), and also plays a role in the

differentiation of endocrine part of the pancreas related to insulin production (Sanchez

et al. 2016).

Valproic acid exposure on certain days of pregnancy would affect the

expression of genes that play a role in the development of pancreatic cells,

particularly beta cells. The ability of valproic acid in influencing the histones

modification, by inhibiting the histone deacetylases (HDAC), may alter gene

transcription normal during the proliferation and differentiation of the fetus cells,

which was initiated by hyperacetylation DNA histone (Ximenes et al., 2012; Kurihara

et al., 2013; Schulpen et al. 2015). Histone acetylation correlates with gene activation

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and histone modifications at the N terminal tail by acetylation or deacetylation, thus

changing the interaction between histones and DNA, and affect regulation of gene

expression (Schulpen et al. 2015). Valproic acid administration, is able to influence

the cell cycle during organogenesis, which plays a role in the regulation and

development of the cells (Berridge, 2014), which is shown by the low concentrations

of DNA and RNA in babies mice from the treatment group than control group, The

results of the babies mice DNA concentration from treatment group showed a

disturbance in the cell replication cycle, including DNA replication that occurs in S

phase and mitotic sister chromatid separation (M phase). After DNA make

improvements, the cells will enter the cell division phase, if the DNA that can not be

repaired, thus the mutation will be fixed to dividing cells so that the cells to be

transformed (Nurhayati & Lusiyanti, 2014).

DNA damage lead to instability of RNA synthesis (Shkreta and Chabot 2015),

to the decrease in the ability of RNA to synthesize proteins. High RNA concentration,

indicating a high protein synthesis activity that is genetically occur on RNA synthesis.

If RNA is synthesized actively, the cell growth is faster. Therefore, the speed of cell

growth is related to the concentration of RNA/DNA ratio, which is a form of

expression of the growth cells character in the tissue. According to Satterwhite (2007)

RNA/DNA ratio is used to see the growth, based on the assumption that the amount of

DNA, the main carrier of genetic information, is stable under changing environmental

situation in the somatic cells of a species, whereas the amount of RNA are directly

involved in protein synthesis, which vary on life stage and size of the organism to

changing environmental conditions. In individuals who have a RNA/DNA ratio

demonstrated a high good growth, while the low RNA/DNA ratio was vice versa

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(Chcharo and Chcharo 2008). According Reef (2010) the growth demonstrated by

the presence of intensive protein synthesis during active growth and cell enlargement,

indicated by a DNA concentration remains constant, but an increase in the RNA/DNA

ratio as a potential indicator of a cell (Chcharo and Chcharo 2008). Babies mice

RNA/DNA ratio from group K0 was higher compared with the mice of the treatment

group P1, P2, and P3. The results showed baby mice from the parent group K0 have

the quality of growth or better development of pancreatic than the treatment group.

VPA administration during pregnancy has the ability to influence the process

of organogenesis insulin-producing beta cells, decreasing the RNA concentration

directly illustrates the reduction in protein synthesis (Olivar et al. 2009). This is

shown by low levels of insulin, resulting in high levels of glucose in mice from

treatment group at gestational day 10, day 13, and day day 16s. During the growth

period, glucose levels increased significantly, although insulin levels did not show

significant differences, but at the end of 32 weeks, insulin levels decreased in all

babies from treatment group. Increased blood glucose levels that previously followed

by a decline, indicating beta cells increase insulin secretion by releasing the proinsulin

for decreased the glucose levels. Increased secretion of proinsulin according Pscherer

et al. (2012), associated with dysfunction and increased beta cell loss.

Hyperproinsulinemia expressed as the damaging effects of beta cells, which are

indicated by hyperglycemia. In long-term studies of secretion insulin abnormalities

with measurement of proinsulin and insulin levels are an independent predictor of the

incidence of type 2 diabetes (Fizelova et al. 2014).

Blood glucose levels are regulated by the hormone insulin that increases

glucose uptake capacity in the tissue thereby lowering blood glucose levels (Ali and

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Padhi 2009). The lack of effective insulin either because their pancreatic beta cell

dysfunction or glucose intake in peripheral tissues, or both, causing the initial disorder

which is located in peripheral tissues (insulin resistance) and then followed by the

pancreatic beta cell dysfunction (Suryono & Yudha 2012). Elevated blood glucose

levels, is a major component that gives the stimulation of insulin-producing beta cells.

Insulin secretion occurs when pancreatic beta cells can take up glucose for glycolysis

and result in ATP. ATP production will cause the closing of K + channels causing

membrane depolarization resulting Ca2+ Chanel opened. Ca2+ chanel opening results in

Ca2+ enter into the pancreatic beta cells that cause the granules containing insulin to

move to the cell membrane and release insulin exocytosis (Tibrani 2009).

Result of observations immunoreactive to glucagon in the alpha cells appeared

a distribution of alpha cells in the babies mice pancreas from treatment group with the

presence of alpha cells in the peripheral parts of the islets of Langerhans. In the babies

mice from treatment group there was a little difference in the number of cells that

immunoreactive to glucagon, showed that the number of alpha cells increases as

compared with the control group. Effendi and Waspadji (2011) suggest that the

disruption of glucose levels showed a significant relationship between volume and

beta cell with pathological conditions. Enlarged diameter caused by the beta cells of

compensatory mechanisms as a result of insulin resistance, the image of

immunohistochemistry showed the beta cells are relatively less dense (beta cell

density reduction) as well as smaller, even punctuated by other non-beta cells,

furthermore showed the distribution of alpha cells that looked expansive and intrusive

to central direction, giving the impression of an increase in the number of alpha cells,

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which appeared in the microscopic feature of babies mice pancreas from day 16

treatment group.

Results of valproic acid administration in the mother rats can inhibit the

process of babies mice pancreatic organogenesis, with a decrease in insulin secretion

and glucose levels. Microscopic observations by immunohistochemistry showed a

lower immunoreactive to insulin by the pancreatic beta cells of mice treated group,

followed by the concentration of DNA, RNA, and the RNA/DNA ratio that showed a

decrease in protein synthesis (insulin) that more apparent with age. This proves the

inhibition process of organogenesis during the growth of beta cells in the babies mice

that born from parent whose given valproic acid in gestational age day 10, day 13, and

day 16s.

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