Chapter 1

Introduction and Genesis of the thesis

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1.1 Non-Communicable Diseases: An Overview

Non-communicable diseases (NCDs) are one of the major health challenges

of the 21st century as they are the leading cause of death worldwide (Global status

report on NCDs, WHO, 2014). The four most common NCDs are cardiovascular

diseases, cancer, chronic respiratory diseases and diabetes (reviewed by Kroll et

al. 2015). The NCD related deaths account for 80% of the disease burden globally

(Haregu et al. 2015). The World Health Organization (WHO) projects that NCDs

will account for more than three quarters of deaths worldwide (The global burden of

disease update, WHO, 2004).

A number of developing countries are struggling with the high prevalence of

NCDs in terms of both mortality and morbidity (reviewed by Kankeu et al. 2013).

According to a global status report on NCDs, about 48% of NCDs occur in low and

middle income countries compared to 28% in high income countries (Global status

report on NCDs, WHO, 2014). A report describing the proportional mortality due to

NCDs in India suggests that approximately 26% of deaths are due to cardiovascular

diseases (NCD country profile, WHO, 2014) (Fig. 1).

Fig. 1: The Proportion of Mortality (% of Total Deaths) due to NCDs in

India, WHO, 2014

Source: Non-communicable Diseases Country Profile, 2014, World Health Organization.

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The rise in the incidence of NCDs in developing countries is attributed to

population ageing and is driven by rapid urbanization and lifestyle changes (reviewed

by Kroll et al. 2015). Tobacco use, excess alcohol consumption, poor diet and lack of

physical activity contribute to the development of NCDs (Thankappan et al. 2010).

The term metabolic syndrome is used to define the clustering of factors that increase

the risk for type 2 diabetes mellitus (T2DM) and cardiovascular diseases.

Cardiometabolic risk factors include high fasting blood glucose, abdominal obesity,

dyslipidemia (increased triglycerides and low density lipoprotein cholesterol (LDL),

lowered high-density lipoprotein cholesterol (HDL)), insulin resistance and high

blood pressure (reviewed by Kaur, 2014; reviewed by Upadhyay, 2015).

Accumulating evidence suggests that specific nutrient deficiency and/ or

excess influence the development of cardiometabolic diseases. The higher incidence

of these diseases in the developing countries has been attributed to the nutrition

transition (reviewed by Popkin, 2002). The industrialized societies are characterized

by increased consumption of saturated fats, trans-fats and omega-6 fatty acids and a

decrease in the intake of omega-3 fatty acids (reviewed by Maire et al. 2002;

reviewed by Simopoulos, 2008). Reports indicate a significant increase in the

consumption of animal fats (egg, pork, poultry, beef, mutton) and saturated fatty acids

obtained from vegetable oils (coconut/ palm oil, butter) in many developing countries,

especially in South Asia (reviewed by Misra et al. 2010; reviewed by Bishwajit,

2015). The adoption of such western dietary pattern accompanied by a reduction in

the physical activity or sedentary lifestyle is reported to contribute to altered lipid

profile, obesity and elevated blood pressure which are major risk factors for metabolic

syndrome (reviewed by Popkin, 2006; reviewed by Pan et al. 2012).

1.2 Association of Nutrition and Cardiometabolic Risk Factors

The association of macronutrient deficiencies and excesses; carbohydrates

(Hauner et al. 2012; Feng et al. 2015), protein (reviewed by Appel, 2003; reviewed by

Rietman et al. 2014), fats (reviewed by Micha and Mozaffarian, 2010; Ebbesson et al.

2015) with hyperlipidemia, insulin resistance and blood pressure are well

documented. Apart from macronutrients, micronutrients have also been shown to

influence cardiometabolic risk factors. Micronutrients include vitamins (vitamin A, D,

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E, K, B; folic acid, vitamin B6 and B12) and trace minerals (zinc, copper, cobalt,

manganese, selenium, iodine). Among various micronutrients, B vitamins are of

particular importance as they influence cardiovascular diseases (reviewed by Friso et

al. 2012; reviewed by Debreceni and Debreceni, 2014). The next section describes the

association of B vitamins like vitamin B12 and folic acid with cardiometabolic risk

factors.

1.2.1 Vitamin B12

Vitamin B12 is an essential water soluble vitamin, critical for haematopoiesis,

cognitive and cardiovascular function (reviewed by Kibirige and Mwebaze, 2013).

Vitamin B12 is found only in animal foods (reviewed by Simpson et al. 2010;

reviewed by Allen, 2012). It acts as a cofactor for the enzyme methionine synthase

which catalyzes the methylation of homocysteine to methionine (reviewed by Grober

et al. 2013). Sub-clinical vitamin B12 deficiency has been reported in patients with

obesity (Baltaci et al. 2013), insulin resistance (Ho et al. 2014), diabetes (reviewed by

Kibirige and Mwebaze, 2013) and in obese hypertensive patients (Karatela and

Sainani, 2009). Obesity in children and adolescents has been shown to be associated

with low vitamin B12 concentration (Pinhas-Hamiel et al. 2006). Further,

hyperhomocysteinemia (elevated plasma homocysteine caused due to vitamin B12

deficiency) is reported to be associated with increased risk for cardiovascular diseases

(Catena et al. 2015; Alihanoglu et al. 2015).

1.2.2 Folic Acid

Folic acid (vitamin B9) is an essential micronutrient, required for one carbon

metabolism, including the remethylation of homocysteine to methionine (reviewed by

Anderson et al. 2012). It is also a key component of DNA synthesis (reviewed by

Crider et al. 2012). A prospective study found that low serum concentrations of folate

are associated with higher incidence of acute coronary events in middle aged men

(Voutilainen et al. 2000). Folate deficiency has been shown to promote oxidative

stress and increases the risk for diabetes and cardiovascular disease in spontaneously

hypertensive rats (Pravenec et al. 2013). Several studies report positive effects of

folate intervention on endothelial function and cardiovascular risk factors (Boushey et

al. 1995; reviewed by Verhaar et al. 2002; Doshi et al. 2002). It has been suggested

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that B vitamins inhibit atherogenesis by decreasing plasma homocysteine levels

through their antioxidant properties and thus can be used to prevent cardiovascular

diseases (reviewed by Debreceni and Debreceni, 2014).

In addition to micronutrients, fatty acids especially long chain polyunsaturated

fatty acids have been implicated in cardiovascular function and is described below.

1.2.3 Long Chain Polyunsaturated Fatty Acids (LCPUFA)

Long chain polyunsaturated fatty acids (LCPUFA) contain more than 20

carbon atoms and two or more double bonds in their hydrocarbon chains. LCPUFA

are distinguished into two key families; omega-3 and omega-6. The biologically

active forms of omega-3 fatty acids are docosahexaenoic acid (DHA; C22:6 omega-3)

and eicosapentaenoic acid (EPA; C20:5 omega-3) (reviewed by Robinson et al. 2010).

A series of studies report altered LCPUFA profile in obesity with a decrease in

omega-3 fatty acids and an increase in saturated fatty acids (Scaglioni et al. 2006;

Karlsson et al. 2006; Verduci et al. 2011; reviewed by Fekete et al. 2015). Reduced

omega-3 fatty acid intake or lower plasma phospholipid content has been shown to be

inversely associated with insulin resistance (Damsgaard et al. 2014) and metabolic

syndrome (Saito et al. 2011; Kim et al. 2016). Increased plasma levels of DHA and

EPA have also been shown to be inversely associated with the risk of atherosclerosis

and heart diseases (reviewed by Holub, 2009). The LCPUFA like omega-3 fatty acids

are suggested to be linked with vitamin B12 and folic acid through one carbon cycle.

1.3 Interaction between Micronutrients (Vitamin B12 and Folic Acid) and

Omega-3 Fatty Acids in the One-Carbon Cycle

The one carbon cycle integrates carbon units from amino acids (including

serine and glycine) and is involved in the biosynthesis of lipids, nucleotides, proteins

and the substrates required for methylation reactions (reviewed by Locasale, 2013). It

is compartmentalized in the cell and occurs primarily within the cytoplasm and the

mitochondria (reviewed by Beaudin et al. 2007). Folic acid and vitamin B12 serve as

coenzymes in the one carbon cycle. The central methylation pathway involves the

methionine cycle where S-adenosyl methionine (SAM) is formed from adenosine

triphosphate (ATP) and methionine by methionine adenosyl transferase enzyme

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(reviewed by Anderson et al. 2012). In turn, SAM is converted to S-adenosyl

homocysteine (SAH) which acts as a precursor for homocysteine. Methionine is

regenerated when a methyl group from 5-methyl tetrahydofolate (5-MTHF) is

transferred to homocysteine. This step of the cycle requires vitamin B12 as a cofactor

for the enzyme methionine synthase. The methionine cycle is associated with the

transsulfuration pathway through the intermediate homocysteine. In this, serine can be

directly metabolized through the transsulfuration pathway that results in the formation

of glutathione (redox regulating system in cell) (reviewed by Locasale, 2013). A

crucial step in the central methylation cycle is where SAM acts as a universal methyl

donor and is converted to SAH. The methyl groups generated from SAM are

transferred to many methyl acceptors such as phospholipids, neurotransmitters and

nucleic acids (RNA, DNA) as shown in Fig. 2.

Fig. 2: Interaction between Vitamin B12, Folic Acid and Omega-3 Fatty Acids in

the One-Carbon Cycle

THF: Tetrahydrofolate; 5,10-MTHF: 5,10-Methylene tetrahydrofolate; 5-MTHF: 5-Methylene

tetrahydrofolate; MS: Methionine synthase; SAM: S-adenosyl methionine; SAH: S-adenosyl

homocysteine; PE-DHA : Phosphatidyl ethanolamine - docosahexanoic acid; PC-DHA: Phosphatidyl

choline - docosahexanoic acid; PEMT- Phosphatidylethanolamine-N-methyl transferase; MT: Methyl

transferases.

Source: Modified from Kulkarni et al. 2011a, PLoS One. 10; 6(3):e17706.

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Phosphatidylethanolamine-N-methyl transferase (PEMT) is a major methyl

group acceptor (Hartz et al. 2006). This enzyme catalyzes the synthesis of

phosphatidyl choline (PC) from phosphatidyl ethanolamine (PE). PEMT prefers

species of PE containing polyunsaturated fatty acids (PUFA), predominantly DHA,

for the synthesis of PC (van Wijk et al. 2012). Thus, PC synthesis by the PEMT

pathway plays a critical role in transport of DHA from the liver to plasma and other

tissues (Watkins et al. 2003).

The methyl groups from SAM are also transferred to DNA. DNA methylation

is considered as an important epigenetic mechanism for the control of gene expression

(reviewed by Anderson et al. 2012). Regulation of gene expression is important

during critical periods of growth and development. Thus, dietary constituents serve as

major determinants of chronic diseases such as obesity, diabetes and cardiovascular

disease (reviewed by Jimenez-Chillaron et al. 2012).

Human studies carried out in our department on pregnant women have

extensively discussed the interaction between folic acid, vitamin B12 and DHA in the

one carbon cycle (Dangat et al. 2010; Kulkarni et al. 2011b, c; Dhobale et al. 2012a,

b). Animal studies in our department have reported that alterations in maternal

vitamin B12 and folic acid status affects LCPUFA levels in the placenta and in the dam

and pup plasma, brain and liver (Roy et al. 2012; Wadhwani et al. 2012; Sable et al.

2012; Meher et al. 2014a, b). It also reduces the expression of delta 5 desaturases in

the dam liver (Wadhwani et al. 2012) and placenta (Wadhwani et al. 2013) and fatty

acid transport proteins (FATP1 and FATP4) in the placenta (Wadhwani et al. 2013).

Further, the deficiency of vitamin B12 and folic acid starting from pre-conception and

continuing throughout pregnancy and lactation has been shown to lower the

expression of peroxisome proliferator activated receptors (PPARγ) in the placenta and

liver of the offspring (Meher et al. 2014a, b). Most of the above adverse effects of

vitamin B12 were partially or completely ameliorated by omega-3 fatty acid

supplementation suggesting that they are interlinked in the one carbon cycle. The next

section describes the effects of maternal micronutrients and the risk for NCDs.

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1.4 Nutrition during Pregnancy and Offspring Health

The nutritional status of the woman, prior to conception and during pregnancy

is recognized as an important determinant of pregnancy outcome. The diet of the

mother during pregnancy is critical for the development and differentiation of fetal

organs (reviewed by Cetin et al. 2010; reviewed by Odent, 2014). Deficiency of

nutrients during pregnancy has adverse consequences on postnatal health of the

offspring (reviewed by Christian and Stewart, 2010; reviewed by Adair, 2014).

Maternal nutrient restriction has been reported to lead to lower birth weight (reviewed

by Molloy et al. 2008; reviewed by Muthayya, 2009), restricted postnatal growth

(Chen et al. 2009), altered organ/ body weight ratios (Watkins et al. 2011) and

congenital malformations (reviewed by Kontic-Vucinic et al. 2006). Several animal

studies have demonstrated that maternal undernutrition before or during pregnancy;

global diet restriction (Ozaki et al. 2001), energy restriction (reviewed by Pico et al.

2012), protein restriction (Cezar de Oliveira et al. 2016) affects fetal growth and

metabolism and thereby increases the risk for insulin resistance, diabetes and

cardiovascular diseases in the offspring in later life (reviewed by Szostak-Wegierek,

2014). Therefore an adequate and balanced supply of both macro and micro-nutrients

is critical for maintaining pregnancy and appropriate fetal growth (reviewed by Rao et

al. 2012).

1.5 Alterations in Maternal Micronutrient Levels in the One Carbon Cycle

and Risk for NCDs in the Offspring

Over recent years, human and animal studies have shown that maternal

micronutrient deficiencies (vitamin B12 and folic acid) increase the risk for diabetes,

obesity and blood pressure in the offspring (Sinclair et al. 2007; Yajnik et al. 2008;

Kumar et al. 2013; Ghosh et al. 2016). It has been proposed that folic acid can

influence lipid metabolism through the one carbon cycle (reviewed by da Silva et al.

2014). However, the role of vitamin B12 and omega-3 fatty acids on lipid metabolism

through the one carbon cycle needs to be explored.

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1.6 Maternal Nutrition and Lipid Metabolism

1.6.1 Lipid Metabolism

Lipid metabolism involves catabolic processes that generate primary

metabolites of fatty acids to produce energy and anabolic processes that form

biologically important molecules from fatty acids and dietary sources. The liver plays

a key role in lipid metabolism (reviewed by Zhou and Liu, 2014). Liver fatty acids

can be derived from the diet and/or are synthesised de novo. Adiponectin, an

adipocytokine is known to regulate the expression of hepatic genes critical for lipid

metabolism (Chen et al. 2013; reviewed by Fu, 2014). Some of these effects are

modulated by AMP-activated protein kinase (AMPK) and acetyl CoA carboxylase

(ACC) activity (Tomas et al. 2002). Fatty acid synthesis is regulated by ACC-1 and

fatty acid synthase (FAS) and these fatty acids are further utilized for the formation of

triglycerides (TG) (reviewed by Shi and Burn, 2004). Glycerol kinase is a

phosphotransferase enzyme involved in TG and glycerophospholipid synthesis (Rahib

et al. 2007). Thus, through lipogenesis and subsequent TG synthesis, energy can be

efficiently stored in the form of fats. Apolipoprotein B (apo B) is a protein involved in

the metabolism of lipids and is the main protein constituent of lipoproteins such as

very low density lipoprotein (VLDL) and low density lipoprotein (LDL) that

transports TG from the liver (Packard et al. 2000). Fatty acids are catabolised in the

mitochondria through the β-oxidation pathway (reviewed by Poirier et al. 2006).

Carnitine palmitoyltransferase-1 (CPT-1) is the enzyme that controls the rate of

transfer of LCPUFA into the mitochondria as shown in Fig. 3.

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Fig. 3: Overview of Fatty Acid Synthesis and Oxidation in the Liver

ADP: Adiponectin, AMPK: 5' Adenosine monophosphate-activated protein kinase, ACC-1: Acetyl

CoA carboxylase; FAS: Fatty acid synthase; CPT: Carnitine palmitoyl transferase, GK: Glycerol

kinase, apo B: Apolipoprotein B, LDL: Low density lipoprotein, VLDL: Very low density lipoprotein,

HMG CoA reductase: 3-Hydroxy-3-methyl-glutaryl-CoA reductase

Source: Modified from Wakil and Abu-Elheiga, 2009, Journal of Lipid Research, 50: S138-43.

1.6.2 Lipid Metabolism during Pregnancy

There are major changes in the maternal lipid metabolism during pregnancy to

ensure a continuous supply of nutrients to the growing fetus. Accumulation of fat in

maternal depots is known to occur during early pregnancy. The lipolytic activity in

the maternal adipose tissue increases during late pregnancy which plays a key role in

the fetal development (reviewed by Herrera and Ortega-Senovilla, 2010). It is well

known that the dietary lipid intake during early pregnancy modulates lipid

metabolism in the fetus (Fernandes et al. 2012). Maternal lipid metabolism has been

shown to be associated with fetal lipids, fetal growth and fat mass (reviewed by

Herrera and Ortega-Senovilla, 2014). A report suggests that the metabolic set points

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of lipid metabolism are determined prenatally and have long-term effects on the adult

offspring (Schindler et al. 2014).

Evidence supports the notion that programming effects are a result of

epigenetic changes (Sookoian et al. 2013). Epigenetic modifications due to nutrient

manipulations are suggested to lead to alterations in the key genes involved in lipid

metabolism during the early periods of development (reviewed by da Silva et al.

2014) and thereby influence lipid metabolic pathways (reviewed by Ferrari et al.

2012). Vitamin B12 is a micronutrient that influences epigenetic changes (reviewed by

Anderson et al. 2012). Epigenetic mechanisms are also modulated by DHA, a major

omega-3 fatty acid with potential impact on the growth and development of the child

(Lee et al. 2014). In view of this, the possible role of maternal micronutrients and

omega-3 fatty acids on fetal lipid metabolism is discussed below.

1.6.2.1 Maternal Vitamin B12 and Lipid Metabolism

Vitamin B12 is a cofactor for the mitochondrial enzyme methyl malonyl CoA

mutase which catalyses the synthesis of succinyl-CoA from methyl malonyl CoA

(MMCoA) (reviewed by Finer et al. 2014). Vitamin B12 deficiency blocks the

synthesis of succinyl-CoA and leads to elevated concentrations of methyl malonic

acid and MMCoA. Increased levels of MMCoA inhibit the activity of CPT-1 (enzyme

involved in fatty acid oxidation) resulting in the inhibition of β-oxidation that leads to

lipid accumulation. Thus, vitamin B12 influences mitochondrial energy and lipid

metabolic pathways (reviewed by Rush et al. 2014).

A recent human study demonstrates that low maternal vitamin B12 status is

inversely associated with the TG levels and homeostatic model assessment- insulin

resistance (HOMA-IR) while it was positively associated with cord blood HDL

cholesterol levels in the offspring (Adaikalakoteswari et al. 2015). Further, low

maternal vitamin B12 status during pregnancy is reported to be associated with obesity

in the offspring in later life (reviewed by Smith et al. 2008; Yajnik et al. 2008).

Maternal vitamin B12 restriction during peri /postnatal period in rats is known to

increase body fat, plasma lipid levels and the activities of lipogenic enzymes, ACC

and FAS and it has been speculated that impaired insulin sensitivity and increased

MMCoA lead to increased lipogenesis (Kumar et al. 2013).

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1.6.2.2 Maternal LCPUFA and Lipid Metabolism

Fatty acids are the precursor molecules for lipid synthesis and early deviations

in the maternal fatty acid metabolism are known to influence neuroendocrine and lipid

metabolic pathways in the fetus (reviewed by Innis, 2011). Literature indicates that

inadequate maternal intake or metabolism of fatty acids increases the risk for

developing metabolic diseases in the offspring in later life (Mennitti et al. 2015).

Reports suggest that maternal omega-3 and omega-6 fatty acid status influences

adiposity in the offspring (Donahue et al. 2011; Moon et al. 2013). A study by de

Vries et al. reports that maternal plasma dihomo-γ-linolenic acid concentration during

pregnancy is associated with increased body mass index (BMI) in the children at 7 yrs

of age (de Vries et al. 2014). Animal studies demonstrate that maternal and post-

weaning diets containing fatty fish or omega-3 fatty acid supplementation improves

lipid profile in the offspring (Hussain et al. 2013; Bremer et al. 2014). Maternal

supplementation with DHA is reported to increase CPT-1 gene expression and

decrease blood lipid in the rat offspring (Gong et al. 2009). In view of the above

literature, the possible mechanisms through which vitamin B12 and omega-3 fatty

acids influence lipid metabolism are discussed below.

A. DHA Enriched Phospholipids and Lipid Metabolism

It has been suggested that dietary B vitamins (folic acid, vitamin B12, B6)

influence the fatty acid profile, particularly DHA by influencing the synthesis of

phospholipids such as PC and PE (van Wijk et al. 2012). The interdependence

between vitamin B12, folic acid and phospholipid DHA concentration has been

reported in studies carried out in humans (Li et al. 2006; Crowe et al. 2008). It has

been reported that PE derivatives in the liver of mice mainly represent

polyunsaturated PE derivatives with PE being a major source of PUFA (Leonardi et

al. 2009). Vitamin B12 deficiency is reported to increase the PEMT activity (Khot et

al. 2014a) while folic acid deficiency is reported to decrease PEMT activity in rats

(Akesson et al. 1982).

The PEMT pathway is required for the regulation of VLDL and its

components in the plasma of mice (Noga et al. 2002). However, the inhibition of

PEMT activity affects incorporation of lipids into VLDL particles in rat liver cells

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(Nishimaki-Mogami et al. 2002) leading to accumulation of hepatic TG (Zhao et al.

2009). A report indicates that the PEMT pathway plays an important role in lipid

metabolism by regulating VLDL secretion and synthesis of PUFA rich in PC,

therefore dysregulation in the PEMT activity can cause changes in the lipid

metabolism (Watkins et al. 2003). It has been suggested that defects in the PEMT

activity and PUFA availability may affect adiponectin which is essential for the

regulation of lipid metabolism.

B. Adiponectin and Lipid Metabolism

Adiponectin is known to play a crucial role in hepatic lipid metabolism

(Tomas et al. 2002; Chen et al. 2013). Studies suggest that adipokines that regulate

hepatic lipid metabolism are epigenetically “programmed” and disturbances of

adiponectin and leptin methylome in the adipose tissue interfere with postnatal growth

and development (Houde et al. 2013). It has been reported that fetal adaptations to a

high fat diet in utero include changes in the fetal hepatic gene expression and

alterations in circulating cytokines may predispose the offspring to metabolic

syndrome in adulthood (Masuyama and Hiramatsu, 2012; Vuguin et al. 2013).

Maternal adipocytokines are also associated with fetal growth (reviewed by Herrera

and Ortega-Senovilla, 2010). Disturbances in the maternal adiponectin system are

associated with diabetes and obesity in the mother (reviewed by Cikos, 2012) but its

consequence in fetal lipid metabolism need to be explored.

Dietary nutrients are known to influence adiponectin levels. Vitamin

restriction during pregnancy is reported to lower plasma adiponectin levels and

increase body fat in the offspring (Lagishetty et al. 2007). Maternal vitamin B12

restriction is reported to lower plasma adiponectin levels in the adult rat offspring

(Kumar et al. 2013). Maternal DHA supplementation has been shown to increase the

adiponectin expression in intrauterine growth restricted (IUGR) rats (Bagley et al.

2013). It has been reported that omega-3 fatty acids act as naturally occurring inducer

of adiponectin (Neschen et al. 2006; Kalupahana et al. 2011). They upregulate

adiponectin gene expression in 3T3-L1adipocytes (Oster et al. 2010) and raise

adiponectin secretion in a dose dependent manner in mice (Neschen et al. 2006).

Long-term supplementation with omega-3 fatty acids is reported to increase plasma

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adiponectin levels and reverse dyslipidemia in sucrose-fed, insulin resistant rats

(Rossi et al. 2005).

C. Altered Chromatin Methylation, Gene Expression and Lipid Metabolism

Epigenetic changes such as chromatin methylation and histone modifications

contribute to fetal metabolic programming (reviewed by Sookoian et al. 2013) and

also affect lipid metabolism pathways (Ferrari et al. 2012). DNA methylation that

occurs during embryonic and fetal development is known to modulate gene

expression, cell differentiation and organ formation (reviewed by Chmurzynska,

2010). Studies suggest that a limited capacity to perform methylation reactions play

an important role in the lipid metabolism (Espe et al. 2010) particularly in lipid

storage patterns (Slow and Garrow, 2006). Impaired methylation capacity promotes

hepatic fat accumulation through an impairment of PC synthesis, a major

phospholipid required for VLDL assembly and homeostasis (reviewed by Obeid and

Herrmann, 2009; reviewed by da Silva et al. 2014).

Maternal periconceptional B vitamin deficiency in sheep is reported to

influence DNA methylation and increase insulin resistance and blood pressure in the

offspring (Sinclair et al. 2007). Feeding high levels of folate, vitamin B12 and choline

during pregnancy is reported to cause epigenetic modifications and result in adiposity

in the mice offspring (Wolff et al. 1998). Deficiency of dietary methyl donors is

reported to lead to metabolic changes in the cell which may be indicated by higher

homocysteine levels, disturbed lipid metabolism and dysregulation of DNA

methylation (reviewed by Obeid, 2013). Methyl donor supplementation has been

shown to modify the DNA methylation profile of the FAS enzyme and reduce fatty

liver in rats fed an obesogenic diet (Cordero et al. 2013).

We have recently reported that inadequacy of vitamin B12 and LCPUFA

containing phospholipids in the one carbon metabolic pathway result in the diversion

of methyl groups towards DNA eventually resulting in aberrant DNA methylation

patterns (reviewed by Khot et al. 2014b). Thus, a low vitamin B12 and omega-3 fatty

acid intake or metabolism during pregnancy may affect DNA methylation patterns

and differentially regulate the expression of genes influencing lipid metabolism.

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An altered lipid metabolism as a consequence of any of the above pathways

leads to accumulation of lipid metabolites in the maternal circulation which may be

transferred to the fetus, impairing fetal lipid metabolism and increasing the risk for

lipid disorders and NCDs in the offspring.

1.7 Genesis of the Thesis

Micronutrient deficiencies which persist over generations are of great concern

throughout the world (reviewed by Bailey et al. 2015) especially in developing

countries like India (reviewed by Vijayaraghavan, 2002; reviewed by Kotecha, 2008).

In recent years, there has been growing interest in vitamin B12 since its deficiency is

prevalent in patients with metabolic disorders (Karatela and Sainani, 2009; reviewed

by Kibirige and Mwebaze, 2013; Baltaci et al. 2013). Vitamin B12 deficiency is

common in India where the prevalence of cardiometabolic disorders is also high

(reviewed by Finer et al. 2014). Reports speculate that the long history of inadequate

vitamin B12 intake in Indians may contribute to the high rate of NCDs (Yajnik et al.

2008). The long-term consequences of vitamin B12 deficiency are not completely

known but are suggested to have adverse effects on cognition, vascular health and

pregnancy outcome (reviewed by O'Leary and Samman, 2010).

Several human and animal studies have demonstrated that a low vitamin B12

status during pregnancy is associated with IUGR (Muthayya et al. 2006b; reviewed by

Sande et al. 2013), low birth weight (Muthayya et al. 2006a), adiposity (Yajnik et al.

2008; Kumar et al. 2013), insulin resistance (Yajnik et al. 2008), low levels of HDL

cholesterol (Adaikalakoteswari et al. 2015) and high blood pressure in the offspring

(Sinclair et al. 2007). In view of this, vegetarian women are being advised to take

vitamin B12 supplementation during pregnancy and lactation to avoid the risk of

vitamin B12 deficiency and improve pregnancy outcome (reviewed by Roed et al.

2009, reviewed by Dror and Allen, 2012). The fortification of flour with vitamin B12

(Winkels et al. 2008; reviewed by Suchdev et al. 2015) or intakes of micronutrient

powders (WHO, 2011) are suggested to serve as effective strategies to improve the

vitamin B12 status in pregnant women, children and older people (reviewed by Allen,

2009).

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There are no reports which have examined the effects of vitamin B12

supplementation during pregnancy and hence it is difficult to evaluate the risks or

benefits (reviewed by Dror and Allen, 2012). Studies examining the effects of vitamin

B12 supplementation during pregnancy are limited (reviewed by Finkelstein et al.

2015). Data relevant to vitamin B12 fortification remain scarce and no country has yet

mandated vitamin B12 fortification (reviewed by Carmel, 2008). Till date, only one

randomized trial has been conducted to examine the effects of vitamin B12

supplementation during pregnancy (Duggan et al. 2014). Moreover, data is also

lacking on the efficacy of vitamin B12 intervention on maternal and child health

outcomes and the potential mechanisms (reviewed by Finkelstein et al. 2015).

Vitamin B supplementation has also been shown to improve heart rate

variability (Sucharita et al. 2012), reduce the risk of cardiovascular diseases in healthy

subjects (Wang et al. 2015) and insulin resistance in patients with metabolic

syndrome (Setola et al. 2004). However, intervention studies are still needed to assess

the efficacy and functional benefits of vitamin B12 fortification (reviewed by Allen,

2009).

Vitamin B12 fortification is suggested to be complicated because of its low

absorption and limited bioavailability (reviewed by Carmel, 2008). Further, the

relationship between B vitamin supplementation and improved pregnancy outcome

may be complicated by gene-nutrient interactions (reviewed by Finnell et al. 2008). It

has been suggested that the bioavailability of a particular nutrient depends on other

nutrients and this should be taken into consideration when planning any

supplementation or fortification strategy (reviewed by Sandstrom, 2001). Reports

suggest that excess vitamin B12 may lead to the accumulation of cyanocobalamin

(reviewed by Carmel, 2008) which is known to be associated with hepatocellular

damage (Ermens et al. 2003). In view of this, studies indicate a need to examine the

effects of long-term supplementation of micronutrients on risk of developing chronic

diseases in adults (Stewart et al. 2009; McKay et al. 2014).

In addition to vitamin B12, low intake of omega-3 fatty acids has also been

reported in the Indian population (Muthayya et al. 2009; reviewed by Misra et al.

2010). Low omega-3 fatty acid levels are associated with increased risk of

cardiometabolic diseases (reviewed by Misra et al. 2010; Bernardi et al. 2013; Kim et

16

al. 2016). The beneficial effects of omega-3 fatty acids, mainly EPA and DHA in

reducing cardiovascular diseases have also been reported (Jain et al. 2015; Sicinska et

al. 2015; Gilbert et al. 2015). In contrast, other studies report no such effects

(Cummings et al. 2010; Amiano et al. 2014).

Omega-3 fatty acids, particularly DHA is known to increase the length of

gestation and is also implicated in fetal growth and development (reviewed by Innis,

2008; Morse, 2012; De Giuseppe et al. 2014). Studies suggest that inadequate levels

of omega-3 fatty acids during the prenatal and postnatal period influences metabolic

diseases (Bernardi et al. 2013), lean mass (Moon et al. 2013) and blood pressure in the

offspring (reviewed by Armitage et al. 2004). The beneficial effects of prenatal

omega-3 fatty acid supplementation in reducing metabolic syndrome markers in the

hamster offspring have been reported (Kasbi-Chadli et al. 2014). However, there are

no reports examining these effects across subsequent generations.

Some human and animal studies report that dietary fish oil supplement is

susceptible to free radical oxidation and increases the oxidative stress (Oarada et al.

2008; Filaire et al. 2010; Walters et al. 2010; Feillet-Coudray et al. 2013). The role of

LCPUFA as a pro- or anti-oxidant is controversial and requires more detailed

investigation (reviewed by Di Nunzio et al. 2011).

Our departmental studies have earlier extensively reported that vitamin B12

and omega-3 fatty acids are interlinked in the one carbon cycle (Dangat et al. 2010;

Kulkarni et al. 2011c, 2011b; Dhobale et al. 2012a; Wadhwani et al. 2014). In view of

the above reports, it is important to assess the long-term effects of micronutrient and

omega-3 fatty acid supplementation across multiple generations on a vitamin B12

deficient/supplemented diet.

1.8 Hypothesis

“A sustained long-term vitamin B12 deficiency across three generations

will adversely affect the lipid metabolism and thereby alter cardiometabolic

variables which may be ameliorated by a combined supplementation of vitamin

B12 and omega-3 fatty acids” (Fig. 4).

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Fig. 4: Possible Mechanism through which Altered Intake/ Metabolism of

Vitamin B12 and Omega-3 Fatty Acids Influences Lipid Metabolism

THF: Tetrahydrofolate; 5,10-MTHF: 5,10-Methylene tetrahydrofolate; 5-MTHF: 5-Methylene

tetrahydrofolate; MS: Methionine synthase; SAM: S-adenosyl methionine; SAH: S-adenosyl

homocysteine; MMCoA: Methyl malonyl CoA; CPT-1: Carnitine palmitoyltransferase; CH3 –

Methyl groups; PE-DHA: Phosphatidyl ethanolamine - docosahexanoic acid; PC-DHA: Phosphatidyl

choline - docosahexanoic acid; PEMT: Phosphatidylethanolamine-N-methyl transferase; LCPUFA:

Long chain polyunsaturated fatty acids; PPAR: Peroxisome proliferator activated receptor; SREBP:

Sterol regulatory element-binding protein; ACC: Acetyl CoA carboxylase, FAS: Fatty acid synthase

Vitamin B12 deficiency increases homocysteine and MMCoA levels. Higher MMCoA inhibits

CPT-1 activity, thereby promoting lipid accumulation. Vitamin B12 deficiency decreases

PEMT activity, synthesis and transport of LCPUFA and affects the key genes (ACC-1, CPT-

1) involved in the lipid metabolism. Low intake/ metabolism of LCPUFA down regulates

adiponectin which subsequently enhances the lipogenesis and lowers β-oxidation.

Inadequacy of LCPUFA containing phospholipids in the one carbon metabolic pathway

diverts the methyl groups towards DNA, resulting into aberrant DNA methylation patterns

which alter the expression of genes involved in the lipid metabolism.

Source: Modified from Khaire et al., 2015, Prostaglandins, Leukotriens and Essential Fatty acids,

98:49-55.

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The present study for the first time investigates the long-term effects of

vitamin B12 deficiency across three generations on cardiometabolic variables and the

possible mechanism influencing lipid metabolism. This study also examines whether

these effects of vitamin B12 deficiency can be ameliorated by a vitamin B12

supplementation / combined supplementation of vitamin B12 and omega-3 fatty acids.

The current work will provide vital clues for supplementation strategies to reduce the

burden of NCDs.