Effect of gestational HDAC-2 inhibition on
cardiometabolic and reproductive functions in offsprings
of STZ-nicotinamide-induced gestational diabetic Wistar
rats
BY
Areola, Emmanuel Damilare
(03/47KB017)
A Ph.D. proposal presentation to the Department of Physiology Faculty of Basic
Medical Sciences, College of Health Sciences, University of Ilorin, Ilorin
Supervisor: Dr. L.S. Ojulari
Outline
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Title
Outline
Literature review
Justification
Hypothesis
Aims and objective
Methodology
Expected outcome
• Benefits and
beneficiaries
• Budget
• Source of funds
• Time-line
• Appreciation
• References
2
Literature review: Diabetes mellitus (DM)
DM is a disease characterized by derangement
in carbohydrate, protein and lipid metabolism
caused by the complete or relative insufficiency
of insulin secretion and/or insulin action
(American Diabetes Association, 2009).
3
Literature review: Insulin resistance
.
Fig.1: Relationship between insulin sensitivity and insulin release in health and disease
(Steven et al., 2006; Nature publishing group)
4
Literature review: Diabetes in pregnancy
• Diabetes in pregnant women is associated with an
increased risk of maternal and neonatal morbidity
and remains a significant medical challenge.
Diabetes during pregnancy may be divided into
clinical diabetes (women previously diagnosed
with type 1 or type 2 diabetes) and gestational
diabetes (Forsbach-Sanchez et al., 2005)
5
Literature review: Gestational Diabetes
(GDM)
• Gestational Diabetes, is defined as any glucose
intolerance detected for the first time during
pregnancy.
– It has evolved from being just a diagnosis associated
with the metabolic risk of type 2 diabetes to a clinical
condition associated with higher risks for maternal
morbidity and fetal deleterious programming and
offspring illness (Forsbach-Sanchez et al., 2005).
6
Literature review: GDM
• The offspring of women who had gestational
diabetes are known to be at risk of diseases
– cardiovascular,
metabolic
and
reproductive
disorders
• due to deleterious intrauterine programming.
• The mechanism of this fetal programming is unclear and
are thought to include epigenetic modifications
7
Literature review: GDM
Fig. 2: Pathway of genetic and epigenetic risks of type 2 diabetes and cardiometabolic
disorders. Khullar et al., 2017
8
Literature review: HDAC
• Histone deacetylase (HDAC) activity is an
important epigenetic process
– HDAC is an enzyme with well-known functions in
the regulation of gene transcription in the nucleus
• HDAC interacts with corepressor proteins to form active
corepressor complexes that catalyzes removal of acetyl
groups from histone proteins to inhibit gene expression
(Guan and Xiong, 2011)
9
Literature review: HDAC
• 18 HDAC enzymes have been identified in
mammalian cells,
• They are divided into 4 classes
• Three of the four classes (Classes I, II, and IV)
are zinc-dependent enzymes while Class III
HDACs are NAD+-dependent.
– Class I HDACs: HDACs(1-3) & HDAC8 are
ubiquitously expressed in the nucleus of cells
– HDACs(1&2) are primarily nuclear while HDAC3 &
HDAC8 can shuttle in and out of the nucleus (Khan
et al., 2008).
10
Literature review: HDAC
• Class II HDACs are associated with tissue
specific functions, and deacetylate many
nonhistone proteins.
• IIA: HDACs (4, 5, 7 & 9)
– Class IIA HDACs show both nuclear and cytosolic
localization, shuttling between these two
compartments in response to different signals
(Khan et al., 2008; Drogaris et al., 2012).
• IIB: HDAC6 and HDAC10. Class IIB HDACs are
localized mainly in the cytoplasm and appear
to function as regulators of signal transduction
and motility (Fournel et al., 2008).
11
Literature review: HDAC
• Class III HDACs, also called sirtuins, consisting of
SIRT 1–7, (Barneda-Zahonero et al., 2012).
– regulate biological functions such as oxidative stress,
DNA repair, metabolism, and aging (Bosch-Presegue
and Vaquero, 2011; Saunders and Verdin, 2007)
• HDAC11 is the only member of Class IV HDACs
Little is known about HDAC11,
– expresson has been noted in the kidney, brain, testes,
heart, and skeletal muscle (Barneda-Zahonero et al.,
2012).
– Shown to regulate oligodendrocyte development (Liu
et al., 2009) and expression of interleukin-10 by
antigen-presenting cells (APCs) (Villagra et al., 2009) 12
Literature review: HDAC
Table 1:
13
Kim and Bae, 2011
Literature review: HDAC
• Recent studies suggested that HDAC exhibits activity
in the cytosol and mitochondria to regulate
acetylation of metabolic enzymes (Guan and Xiong et
al., 2011) as a form of post translational modification.
• More than 20% of mitochondria proteins are
regulated by acetylation (Kim et al., 2006; Choudhary
et al., 2009).
• Presently, regulation of HDAC activity is a new
approach to modify glucose and fatty acid metabolism
in the treatment of type 2 diabetes.
14
Literature review: HDAC
• Studies have found significant linkage
between the chromosomal region 6q21,
where HDAC2 is located, and both T1D
and T2D (Nerup and Pociot, 2001.)
– Indicating that HDAC2 could play a role in diabetes
mellitus.
15
Literature review: HDACi
• A large number of HDAC inhibitors have
been purified from natural sources, or
have been synthesized.
16
Literature review: Classes of HDACi
Fig. 3: Structure of HDAC inhibitors Kim and Bae, 2011
17
Literature review: Classes of HDACi
Fig. 4: Structure of HDAC inhibitors Kim and Bae, 2011
18
Literature review: HDACi
• Histone deacetylase inhibitors (HDACi)
show
promising
properties,
as
antiinflammatory
demonstrated
in
an
increasing number of animal and cellular
models of inflammatory diseases (Halili et
al., 2009).
19
Justification
• Gestational diabetes mellitus (GDM)
• the most common metabolic disorder of pregnancy.
• As at 2015,
• worldwide prevailence was between 1 and 45% of
pregnancies (Agarwal et al., 2015)
• as at 2021,
• worldwide prevalence is 16.9% in women of child
bearing age between 20 to 49 years old (Chai et al.,
2021)
Justification
• Studies have shown that patients with GDM are at risk
of developing type 2 diabetes later in life or after
delivery (Alejandro et al., 2020)
• Also the children born by gestational diabetes-affected
pregnancies are at risk of postnatal disorders
– macrosomia, neonatal hypoglycemia, respiratory distress
syndrome (Ley et al., 2020) and future metabolic syndrome
and obesity-related disease
Justification
• HDACs have been recently shown to be involved in
regulating gene expression of several key molecules
involved in microvascular complication of diabetes
(Portela and Esteller, 2013).
• There is increased expression of HDAC2 mRNA and
HDAC1/2 deacetylase activity in hearts from diabetic
rats (Cox and Marsh, 2013)
Hypothesis
• Gestational
maternal
HDAC2
inhibition
ameliorates cardiometabolic and reproductive
dysfunction in offspring of gestational diabetic
Wistar rats.
23
Aim of study
• To investigate the effect of gestational HDAC-2
inhibition on cardiometabolic and reproductive
functions in the offspring of gestational type 2
diabetic Wistar rats.
24
Objectives
• The objectives of this study are to determine the effect
of gestational HDAC-2 inhibition on
– Maternal cardiometabolic status and fetal outcome at term
– Male and female offspring cardiometabolic status after 5
weeks of postnatal life
– Male and female offspring reproductive status after 5 weeks
of postnatal life
25
Methodology
Ethics Statement
• All the animals to be used in this study shall be given humane
care and the experimental protocols shall be in accordance with
the ethical principles of the University of Ilorin, Ilorin.
Animals
•
Female Wistar rats of about eight weeks will be mated with
mature
males
to
achieve
pregnancy
after
one
week
acclamatization having considered their estrous cycle.
26
Methodology
Induction of GDM
• Gestational diabetes will be induced on gestational day 0
with streptozotocin and nicotinamide. Nicotinamide (120
mg/kg
body
weight;
intraperitonealy)
will
be
administered 15 minutes before administration of STZ (35
mg/kg body weight in 0.1 Molar citrate buffer (pH 4.5)
(Abdul Aziz et al., 2016).
• Treatment with HDAC2 inhibitor (romidepsin; 3 mg/kg)
for 19 days from gestational day 2 to gestational day 20
27
Methodology: Experimental Design
Experimental Design
• The experiment shall be divided into two phases (1
and 2).
– Phase 1 (To determine the role of HDAC-2 in fetal
outcome and cardiometabolic status of gestational
diabetic Wistar rats)
– Animals
• In phase 1, 36 female Wistar rats made pregnant as described
above. The pregnant animals will be randomly sorted into 6
groups (n=6) and treated appropriately as described below
28
Methodology: Experimental design
Phase 1 groups and treatment
•
Group 1 (control) - receive distilled water
•
Group 2 (Diabetic) – receive STZ (35 mg/kg body weight) and Nicotinamide (120
mg/kg body weight)
•
Group 3 (Diabetic + HDAC2i) – receive STZ (35 mg/kg body weight) and
Nicotinamide (120 mg/kg body weight) + (romidepsin, 3 mg/kg) (Gloucester
Pharmaceuticals, Inc.)
•
Group 4 (Diabetic + standard drug) – receive STZ and Nicotinamide (120 mg/kg
body weight)+ metformin (200 mg/kg body weight)
•
Group 5 (HDAC2 inhibitor only) – receive HDAC2i (romidepsin, 3 mg/kg)
(Gloucester Pharmaceuticals, Inc.)
•
Group 6 (metformin only) – receive metformin (200 mg/kg body weight)
29
Methodology: Treatment
• Experimental Design
– Treatment pattern
7 days
Acclimatization;
without treatment
Induction
of GDM
1
Gestational days
Start
21
Sacrifice
HDAC inhibitor treatment
Random rat chow
with water
Monitored rat chow and water
Fig 5: Treatment pattern
30
Methodology: Experimental design
Phase 2 groups and treatment
• The aim of this phase is to determine the role of maternal gestational
HDAC-2 alterations in gestational diabetic Wistar rats offspring
cardiometabolic and reproductive functions
• Grouping and treatment (no treatment)
• Group 1 (phase 1 control offspring)
• Group 2 (phase 1 Diabetic offspring)
• Group 3 (phase 1 Diabetic + HDAC2i offspring)
• Group 4 (phase 1 Diabetic + standard drug offspring)
• Group 5 (phase 1 HDAC2 inhibitor only offspring)
• Group 6 (phase 1 metformin only offspring)
31
Methodology: Measurements
• Body weight and Organ weight
– Body weight of the pregnant animals and offspring shall be taken once in a
week by a weighing scale throughout the experimental period.
– The maternal heart, liver, kidney, visceral fats (pericardial fat, peri vascular fataortic and perirenal fats), pancreas and placenta weights shall be taken.
Likewise, the fetal weight and number shall also be taken after sacrifice.
• Food and Water Intake
– Food and water intake of the pregnant animals and offspring shall be
monitored daily during the experimental period by deducting the weight of
the remaining feed/water from the actual feed/water given to the rats the
previous day. The feed will be weighed on a sensitive weighing scale while the
water is measured in a measuring cylinder.
32
Methodology: Measurements
• Oral Glucose Tolerance Test (OGTT)
• Twenty-four hours before sacrifice, After 12 hrs of fasting, rats
will be given an oral bolus of glucose (2.0 g/kg), and blood
samples will be obtained through the tail at 0, 30, 60, 90 and
120 min after glucose administration to test for blood glucose
level using a glucometer.
• Insulin Resistance
• Homeostatic model assessment of insulin resistance (HOMA-
IR) as an index of insulin resistance as calculated by the
following formula: Insulin (µU) x Glucose (µmol L-1) / 22.5
(Bergman et al., 2003).
33
Methodology: Measurements
• Sacrifice
• At the end of the treatment periods in both phases, rats will
be
anaesthetized
and
euthanized
using
sodium
pentobarbitone (50 mg/kg).
• Pregnant animals and offspring blood samples will be
collected by cardiac puncture, centrifuged at 5,000rpm for 5
minutes for plasma extraction, storage, and subsequent
biochemical analysis.
34
Methodology: Measurements
• Sacrifice
• Pregnant animals and offspring hearts and kidneys and
offspring testes and ovary will be excised for biochemical and
histological analysis, weighed and adjusted for body weight.
The excised organs will be homogenized and in phosphate
buffer solution (PBS) and centrifuged at 3000 rpm for 10
minutes. The supernatant shall be kept for biochemical
analysis.
35
Methodology: Measurements
• Biochemical Analysis
• Blood and tissue samples collected in heparinised bottle will
be centrifuged to separate the plasma which will be used for
biochemical analysis using ELISA kits (Elab science). The
following parameters will be estimated; insulin level, creactive protein, c-peptide, (GS), malonaldehyde (MDA),
interleukin-6 and interleukin-1B, Tissue necrosis factor
alpha, testosterone, estrogen, leutinizing hormone level and
follicle stimulating hormone level.
36
Methodology: Measurements
• Lipid and Lipoprotein estimation
• Blood and tissue total cholesterol (TC), triglyceride (TG), high
density lipoprotein cholesterol (HDL-C) levels will be
determined by standard colorimetric method.
• TC/HDL-C, TG/HDL-C will be calculated
• Uric acid Pathway
• Maternal and offspring Cardiac and hepatic Uric acid and
xanthine oxidase activity will be assessed by standard
colorimetry.
37
Methodology: Measurements
• Western blot Analysis
• Maternal cardiac tissue western blot of total and acetylated
histone 3 (H3) and maternal and offspring cardiac and serum
tumor necrosis factor-alpha and transforming growth factor
beta 1 shall be performed using standard protocol as
described by Li et al., 2011.
38
Methodology: Measurements
• Reproductive analysis
– Full semen analysis, Ovarian histological
analysis (H &E staining)
39
Methodology: statistical analysis
Data will be analyzed via one way ANOVA followed by
the Bonferroni’s multiple comparison tests for analysis of
biochemical data using SPSS version 20.
Significance will be accepted at P <0.05 and data will be
presented as means±SEM unless otherwise stated.
40
Expected outcome
• At the end of this experiment, the study
will provide explanation on the following.
– Role of HDAC 2, an epigenetic molecule in onset and
progression of gestational diabetes
– Role of HDAC2 in fetal outcome within gestational
diabetes milieu
– Role of HDAC 2 in fetal programming for
cardiometabolic and reproductive derangements within
gestational diabetes milieu
41
Benefits and Beneficiaries
• Benefits
– Elucidate further on the cardiometabolic and reproductive impact
of intra-uterine programming in gestational diabetes mellitus.
– To provide information on HDAC2 as a target molecule in treating
gestational diabetes and preventing the associated deleterious
fetal programming.
• Beneficiaries
– University community.
– Individuals with gestational diabetes mellitus or who are
susceptible to it.
– Pharmaceutical companies.
42
Budget
Table 2: Budget
Items
Cost implications (N)
Rats
100,000
HDAC2 inhibitor
80,000
STZ
120,000
OGTT
50,000
Nicotinamide
50,000
Biochemical Kits
950,000
Preparation for thesis
Western Blotting
TOTAL
20,000
144,000
2,774,000
43
Time-line
Table 3: Timeline
October
– September 2017
October 2021
– 2016
September
2022
Activities
Oct
Nov
Dec
Jan
Feb
Mar
Apr
May
Jun
Jul
Aug
Sep
Jun
Jul
Aug
Sep
Survey of Literature
Ethical approval
Gathering of Data
Presentation
Laboratory
experiment
Data analysis
Presentation 2
October 2017– September 2018
October 2022
– September 2023
Activities
Oct
Nov
Dec
Jan
Feb
Mar
Apr
May
Second phase
experiment
Presentation 3
Articles
Conference
Final defense
and
44
Source of funds
• Grants.
• Personal Income.
• Family and friends.
45
Mode of dissemination
• Internal seminars.
• Publications in local and international
journals.
• Conference presentations.
46
Acknowledgements
• My sincere appreciation goes to the following
people
– The Almighty God
– My supervisor; Dr. L.S. Ojulari
– The Head, Department of Physiology
– The post graduate coordinator
– Other postgraduates teachers
– Staff of the Department
– Family and friends
47
References
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Complications, 29 (4), 544 – 549
Alejandro, E.U., Mamerto, T.P., Chung, G., et al. (2020). Gestational diabetes mellitus: a harbinger of the vicious
cycle of diabetes, International Journal of Molecular Sciences, 21(14) 5003.
American Diabetes Association (2009). Diagnosis and classification of diabetes mellitus. Diabetes Care,
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Bosch-Presegue, L. & Vaquero, A. (2011). The dual role of sirtuins in ´ cancer, Genes and Cancer, 2 (6), 648–662.
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Choudhary, C., Kumar, C., Gnad, F. et al. (2009). Lysine acetylation targets protein complexes and co-regulates
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Thank you for listening
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