EVIDENCE FOR AN INFLAMMATORY PATHWAY EXPLAINING THE RELATIONSHIP

BETWEEN BREASTFEEDING AND DECREASED RISK OF TYPE 2 DIABETES

By
Brenna Alvarez

A Senior Project submitted

In partial fulfillment of the requirements for the degree of
Bachelor of Science in Nutrition

Food Science and Nutrition Department

California Polytechnic State University
San Luis Obispo, CA

June 2017



 Abstract
The microbiome has been the focus of recent studies due to its role in healthy and immunity.
There are around 10 times as many bacterial cells as there are human cells. These microbial cells
make up the human microbiome. The gut microbiome has been seen to protect against
pathogens, support intestinal and immune growth and development, and have metabolic
functions. A strong association is apparent between the gut microbiome, inflammation, and type
2 diabetes mellitus (T2DM). T2DM is a metabolic disease characterized by low-grade chronic
inflammation. One of the most important contributors to the development of a healthy and
balanced gut microbiome is breastfeeding. Abnormal microbiota (microbial dysbiosis)
commonly seen in formula fed infants appears to be trademark of T2DM. Immune promoting
constituents in breast milk facilitate infant microbial development and maturation alongside the
development of the immune system. Evidence reviewed suggests that the mechanism behind
increased inflammation seen in T2DM is controlled by the gut microbiome. The studies looked
at involved breastfeedings influence on the microbiome development and the microbiomes
influence intestinal permeability, an increased prevalence of Gram-negative bacteria in the
lumen, increased cytokine production by activated NFkB transcription, amplified innate
inflammatory responses from bacterial translocation, and tissues apoptosis that leads to poor
glucose homeostasis, decreased insulin secretion, and insulin resistance seen in T2DM. There are
no studies that look at the link between breastfeeding, the gut microbiome, and development of
T2DM, but there sufficient evidence exists to suggest the link between all three.
Introduction
According to the American Diabetes Association (ADA), 29.1 million Americans or

9.3% of the population had diabetes in 2012. Of those diagnosed with diabetes, 27.85 million or

95.7% of the population had type 2 diabetes. The incidence of type 2 diabetes mellitus (T2DM)

has dramatically increased over the last couple decades, affecting millions of people worldwide.

This epidemic sparked an ongoing investigation into the etiology of the disease. T2DM is a

metabolic disorder characterized by hyperglycemia, insulin resistance, decreased insulin

production, and inflammation. Low-grade inflammation is prevalent during the development and

onset of the disease. There is growing evidence that low-grade inflammation is a potential

pathway in the pathogenesis of T2DM due to a rise in circulating inflammatory cytokines in the

body.

An altered microbiota plays a key role in the development of inflammation. One of the

most important contributors to the development of a healthy and balanced gut microbiome is

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breastfeeding. Breast milk and its protective immune components have been shown to initiate the

development of the infants innate and adaptive immunity, and recent epidemiological studies

have associated breastfeeding with a reduced incidence of T2DM and other diseases. The

balance of factors in breast milk supports the infants immune system and reduces inflammatory

response. Breastfeeding has been seen to affect the development of the gut microbiome, which in

turn plays a key role in reduced inflammatory response related to breastfeeding. Although

knowledge of the microbiome and its affect on overall health has improved within the last

decade, the microbiome has proven its existence and effects for centuries from ancient cultures

and practices, such as natural childbirth (NC) and breastfeeding (BF). Recent technologies have

replaced these ancient innate practices, producing beneficial results along with unintended

consequences. Such technological developments include Caesarian births, increased sanitization

in birth practices and ways of living, vaccines, and formula feeding. These advancements that

were intended to improve the health of children, reduce pregnancy and childbirth risks, and

benefit infant development have negatively affected the human-microbiome development with

consequent immune dysfunction.

Abnormal microbiota and a reduced complexity of the gut microbial ecosystem

commonly seen in formula-fed infants appear to be trademarks of type 2 diabetes. A strong

association is apparent between the gut microbiome and the onset of T2DM (Karlsson F et al,

2013; Egshatyan L et al, 2016). Formula feeding results in a poorly developed gut microbiome

characterized by high levels of Clostridia, Enterococci, and Enterobacteria. This can affect

GALT development and lead to gut permeability and bacterial translocation. Early colonization

of the infant gut microbiome is one of the main factors contributing to microbial composition

that can lead to the presence or absence of dysbiosis, immune dysfunction, and inflammation.

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Formula feeding can potentially result in an altered microbiome development, resulting in

microbial dysbiosis. Studies show that FF infants have higher levels of gram-negative bacteria

including Clostridia, Enterococci, and Enterobacteria and lower levels of gram-positive bacteria

including Lactobacillus, and Bifidobacteria compared to BF infants (Harmsen et al, 2000;

Bezirtzoglou et al., 2006; Magne et al., 2006; Palmer et al., 2007; Penders et al., 2014). It is

hypothesized in this review that the microbiome mediates the relationship between breastfeeding

and type 2 diabetes through mitigating the inflammation response.

Type 2 Diabetes
The incidence of type 2 diabetes mellitus (T2DM) has dramatically increased over the

last two decades, affecting millions of people worldwide. The cost of diabetes in 2012 was

approximately $245 billion in medical costs and lost work and wages according to American

Diabetes Association (ADA) (American Diabetes Association, 2013). The three most common

diabetes diagnoses are T2DM, type 1 diabetes mellitus (T1DM), and gestational diabetes

mellitus (GDM), along with a multitude of cases of prediabetes. According to the 2014 National

Diabetes Statistics Report by the Centers for Disease Control and Prevention (CDC), 29.1

million Americans or 9.3% of the population had diabetes in 2012, about 1 out of every 11

people. The population of Americans with diabetes increased by 3.3 million from 2010 to 2012

(Figure 1). Approximately 1.4 million Americans are diagnosed each year with diabetes. Of the

estimated 29.1 million with diabetes, 21.0 million were diagnosed and 8.1 million were

undiagnosed. About 1 out of every 4 Americans are unaware of having T2DM. A high

percentage of the 29.1 million Americans with diabetes had T2DM, at 95.7% or 27.85 million

(diagnosed and undiagnosed). In 2012, 86 million or 27.5% of the population age 20 and older

had prediabetes, an increase of 7 million individuals since 2010. This means that 1 out of every 3

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adults has prediabetes. The International Diabetes Federation predicts a 55% increase in the

global prevalence of T2DM by 2035 (Guariguata L et al, 2014). (American Diabetes

Association, 2015; Centers for Disease Control and Prevention, 2014).

Figure 1: Diabetes Statistics in 2010 vs 2012

(ADAs Statistics About Diabetes webpage; from the CDCs 2014 National Diabetes Statistics Report)

T2D was previously referred to as non-insulin-dependent diabetes or adult-onset

diabetes. Type 1 diabetes most commonly develops due to an autoimmune destruction of

pancreatic beta cells that results in the bodys inability to produce insulin. This leads to higher

than normal levels of glucose in the blood, referred to as hyperglycemia. Hyperglycemia is also

present in individuals with T2DM, whereas during the development of T2DM there is a slow

progression of hyperglycemia that eventually leads to insulin resistance. The early progression of

type 2 diabetes is often asymptomatic and can go undiagnosed for many years due to a gradual

increase of blood glucose levels and the development of insulin resistance overtime. This may

explain why common symptoms of T2DM can take a while to occur or go unnoticed, including

frequent urination (polyuria), increased thirst (polydipsia), fatigue, still feeling hungry after a

meal (polyphagia), and slow wound healing. Complications and comorbid diseases that can

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transpire with T2DM include diabetic retinopathy, kidney toxicity, atherosclerosis, and

hypertension (Zhang Y and Zhang H, 2013). Low-grade inflammation is prevalent during the

development and onset of the disease. An altered microbiota plays a key role in the development

of inflammation, the comorbid diseases mentioned above, and T2D.

Etiology and Risk Factors

In people with T2DM, the body becomes resistant to insulin. Insulin is a peptide hormone

produced by the beta-cells of the pancreatic islets in response to increased postprandial blood

glucose levels. Insulin plays a major role in metabolism and the bodys use of digested food for

energy. Glucose is absorbed into the bloodstream from the break down of carbohydrates. After a

meal, insulin signals muscle, fat, and hepatic (liver) cells to absorb glucose from the blood in

order to be used for energy or it signals the liver and muscle tissue to store excess glucose as

glycogen. In a healthy person, the tissues properly respond to insulin and the body is able to

maintain normal blood glucose levels. During the onset of T2D, tissues slowly stop responding

to insulin, leading to insulin resistance. Insulin resistance begins with frequent reoccurring states

of hyperglycemia (higher than normal levels of blood glucose) causing the pancreas to work

harder and produce extra insulin (hyperinsulinemia). Insulin resistance is essentially the result of

hyperinsulinemia. The pancreatic beta cells eventually may no longer be able to keep up with the

bodys increased demand for insulin and insulin production comes to a halt (NIDDK, 2016).

There are several factors that contribute to the development and onset of T2DM in an

individual, including physical activity, diet, excess weight, age, genetic predisposition, and

family history. According to the National Institute of Diabetes and Digestive and Kidney

Disease, these risk factors factors seem to be linked together, and those with multiple known risk

factors are at higher risk of developing the disease (NIDDK, 2016). Obesity is one of the most

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common risk factors of T2DM. Insulin resistance is commonly seen in individuals who have

excess abdominal fat. Lack of physical activity has also been linked to insulin resistance. The

level of physical activity is correlated with weight gain and obesity, which can lead to insulin

resistance. Increased exercise is one of the most effective, and cost-effective, treatments of

T2DM and can significantly reduce insulin resistance (Esteghamati et al., 2009; Sarvas et al.,

2014). A combination of extra weight, fat cells, sedentary lifestyle, and hyperglycemia can

damage the insulin-producing beta cells in the pancreas. This can result in abnormal insulin

levels in the blood. Muscle, liver, and fat cells eventually stop responding to insulin and become

insulin resistant, which causes the pancreas to produce more insulin until the beta-cells become

non-functional. The combination of sedentary lifestyle, diet, and weight gain seem to be the three

most common risk factors that interact with each other during the developmental stage of T2DM.

Along with these risk factors, an increased occurrence of inflammation in the body also plays an

important role in the development of abnormally high blood glucose levels, reduced insulin

secretion, and insulin resistance.

Inflammation in T2DM

There is growing evidence that low-grade inflammation is a potential pathway in the

pathogenesis of T2DM due to a rise in circulating inflammatory cytokines in the body.

Inflammation measurement is a useful predictor of T2DM risk. During the prolonged

progression of T2DM, elevated levels of inflammatory cytokines coincide with constant high

blood glucose levels and with a decline in pancreatic insulin production. The three most common

tests when diagnosing T2DM are measuring levels of hemoglobin A1c (HbA1c), fasting and

postprandial glucose levels, and C-reactive protein (CRP) in the blood. Based on the American

Diabetes Association (ADA), T2DM diagnosis requires a fasting blood glucose level of

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126mg/dL, postprandial glucose 200 mg/dL, HbA1c of 6.5% or higher, CRP is the most

commonly tested inflammatory marker in the body. It is a positive acute-phase protein produced

by the liver that increases in response to inflammation. Research shows that CRP significantly

and positively correlates with insulin resistance and CRP concentration is higher in individuals

with T2DM compared to those without diabetes. Chronic low-grade inflammation in T2DM is

evidenced by elevated levels of CRP in the blood (Freeman et al., 2002; Mahajan et al., 2009;

Sato et al., 2014).

The association between inflammation and T2DM, as well as obesity, has been the focus

of many studies over the past 10 years. Evidence by Freeman et al., 2002 indicates that CRP was

a strong predictor of diabetes development independent of glucose concentrations and other

clinical predictors (HR 1.30; 95% CI 1.07-1.58; P=0.0075). Of the men that were classified as

having T2DM, those in the top quintile with a CRP of 4.18 mg/L had a threefold risk of

developing T2DM compared to those in the lowest quintile with a CRP of 0.66 mg/L (95% CI

1.205.04) (Freeman et al., 2002). The cross-sectional study by Mahajan A et al., 2009 showed

that the 2420 T2DM subjects had higher median high-sensitivity CRP (hsCRP) levels compared

to the 1110 nondiabetic subjects (P<0.0001). After adjusting for age, sex, BMI, HbA1c, and

obesity, high CRP levels were positively associated with T2DM (OR : 1.66; 95% CI: 1.21-2.28;

P=0.002). Diabetic subjects had median hsCRP levels of 1.58 mg/L for males and 2.68 mg/L for

females. Non-diabetic subjects had median hsCRP levels of 1.11 for males and 1.33 for females.

(Mahajan et al., 2009). Sat J et al., 2014 also found that hsCRP was significantly higher in

individuals with T2DM compared to those without diabetes or elevated inflammatory markers

(Sato et al., 2014). These studies have clinical potential toward better predicting individuals at

higher risk of developing T2DM based on elevated inflammatory markers and CRP levels

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(Freeman et al., 2002; Mahajan et al., 2009; Sato et al., 2014). The gut microbiome is an

important contributor to the pathogenesis of T2DM through specific processes that lead to

chronic low-grade inflammation, and the gut microbiome development is strongly influenced by

infant feeding.

Breastfeeding

Human Milk Composition & Health Benefits

Scientists have still not been able to replicate what nature has perfected: breast milk.

Breast milk provides insight into complete nourishment and a protective diet in the investigation

of food and nutrition. Milk synthesis and its adaptive properties vary among individuals. Not

only does human milk comprise various health promoting bioactive compounds and factors, it is

unique and individualized and its composition fluctuates during each feeding and as the infant

ages. According to research, fat content and other live cells and bioactive compounds increase

during the course of a feeding (Khan, 2012). Breastfeeding is dynamic and its composition

continually changes according to the infants needs, unlike infant formula. Infant formula

delivers the same amount of nutrients each feeding. In terms of carbohydrate content, breast milk

contains lactose along with over 130 different oligosaccharides referred to as human milk

oligosaccharides (HMOs), and formula only contains lactose as its carbohydrate source. The

nutritional, developmental, and immunological benefits that these HMOs provide to the infant

are unparalleled. Milk removal during breastfeeding also stimulates gene expression in the

mother, in turn signaling the body to actively secrete a specific amount of lipids, protein,

carbohydrates, and other factors into the milk. The transfer of milk from mother to infant is a

system that actively guides the development of the immune system, metabolic systems, and the

microbiome within the infant (Khan, 2012; Field C, 2005).

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 In addition to providing nutrients and energy, breast milk and its protective immune

components have been shown to initiate the development of the infants intestinal tract and

innate and adaptive immunity. Recent epidemiological studies have associated breastfeeding

with a reduced incidence of gastrointestinal and respiratory infections, atopic diseases, and

immune-mediated and inflammatory diseases (Field C, 2005). Immune promoting constituents in

breast milk facilitate infant immune development and maturation. These constituents are

comprised of leukocytes, macrophages, neutrophils. lymphocytes (white blood cells), cytokines,

growth factors (i.e. epidermal and insulin-like growth factors), hormones, partially digested milk

peptides, long-chain polyunsaturated fatty acids (LCPs), including n-6 (linoleic acid) and n-3

(alpha-linolenic acid), nucleotides, microflora, and bacterial antigens. Also, in recent years,

scientists have found that human breast milk has a much greater concentration of HMOs than

any other mammalian milk. (Table 1) (Field, 2005).

Table 1: Compounds with Immunological Properties in Human Milk

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Breastfeeding & Microbiome Development

The balance of factors in breast milk supports the infants immune system and reduces

inflammatory responses. Breastfeeding has been seen to affect the development of the gut

microbiome, which in turn plays a key role in reduced inflammatory response related to

breastfeeding. Human milk from healthy mothers has around 1 billion microbes per liter. The

bacteria most commonly found from the nipple, surrounding skin, and from the milk ducts in the

breast include Staphylococci, Streptococci, Lactobacilli, Propionibacteria, and Bifidobacteria

(West et al., 1979). After one week of age, Bifidobacteria dominate the infant gut (Harmsen et

al, 2000; Bezirtzoglou et al., 2006; Magne et al., 2006; Palmer et al., 2007; Penders et al., 2014).

The prevalence of Bifidobacteria in the infant gut is due to the HMOs that are digested

and utilized by Bifidobacterium longus infantis (B. infantis) (Sela and Mills, 2010). These

indigestible HMOs are the only human milk factors that dont directly nourish the infant, but are

instead utilized by B. infantis. This particular strain seems to thrive in the presence of HMOs and

produces important compounds called short-chain fatty acids (SCFA) after fermenting these

indigestible HMOs. Formula tries to mimic these oligosaccharides, but they dont seem to

reproduce the same immune capacity or support the growth of the same infantis subspecies of

Bifidobacterium compared to breast milk. Figure 2 below illustrates the difference of gut

microbiota found in breast fed (BF) versus formula fed (FF) infants. HMOs select for specific

infant gut microbial growth. Infant formula, which only contains lactose, is non identical to

breast milk and reduces the exclusivity of the gut microbiota (Selma and Mills, 2010; Yong,

2016).

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 Figure 2: Microbiota found in the guts of breast milk vs formula fed infants
In the BF infant (left), Lactobacillus lactis, Bacteroides fragilis, Staphylococcus
epidermidis, Bifidobacterium bifidum, and Streptococcus pyrogenes are shown.
In the BF infant (right), that is a greater amount of gram-negative bacteria.
Enterococcus faecalis, Escherichia coli, Verrucomicrobium sp., Shigella
dysenteriae, Peptostreptococcus spp, Bacteroides fragilis, Bacilus subtilis, and
Atopobium vaginae are shown in FF infant. Purple= Proteobacteria. Blue=
Firmicutes. Red=Actinobacteria. Green= Bacteroidetes

Breastfeeding & Type 2 Diabetes

Breastfeedings potential to lower the risk of developing T2DM later in life is illuminated

by various beneficial pathways. Research suggests that breastfeeding protects individuals from

T2DM. There is sufficient scientific evidence suggesting that individuals who were formula fed

as infants are more likely to be overweight and develop T2DM later in life (Horta, 2015; Pereira,

2014; Veena, 2011; Mayer-Davis, 2008; Owen, 2006; Lawlor, 2005). It was found that

breastfeeding decreases the risk of developing T2DM by 40% (Owen, 2006). In formula fed

infants, weight gain and an altered development of the gut microbiome appear to mediate the

relationship between infant feeding and risk of T2DM. One avenue that explains breastfeedings

ability to reduce the risk of T2DM is its ability to support gram-positive bacterial growth in the

infant gut, thus promoting a healthy, balanced gut microbial development (gram-positive bacteria

will be described in more detail in the Microbiome section). One particular gram-positive

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bacteria of interest in this paper is B. infantis. Other pathways that connect breastfeeding and its

effect on reducing the risk of T2DM will be described in further detail in later sections

(Ewaschuk et al., 2008; Yong, 2016).

B. infantis is considered a gram-positive bacteria, which are known to have anti-

inflammatory effects. B. infantis has the ability to reduce the risk of inflammation by decreasing

intestinal permeability and inhibiting the production of pro-inflammatory factors in the intestinal

lining including tumor necrosis factor- (TNF-), lipopolysaccharides (LPS), and other pro-

inflammatory markers. These mechanisms help protect against low-grade inflammation seen in

T2DM. Breast milk is the only source of infant nutrition able to unlock the full beneficial

potential of gram-positive bacteria and their ability to flourish in the infant gut (Yong E, 2016).

Research indicates that BF infants have greater levels of gram-positive bacteria in the gut

compared to FF infants, which provides increased protection against developing T2DM. It is

hypothesized in this review that the microbiome mediates the relationship between breastfeeding

and T2DM by reducing the risk of gut microbial dysbiosis, intestinal permeability, and low grade

inflammation as seen in T2DM (Ewaschuk et al., 2008; Yong, 2016).

Microbiome
Description, Structure, Function, & Metabolites

There are around 10 times as many bacterial (or microbial) cells as there are human cells.

These microbial cells make up the human microbiome, defined by the National Institutes of

Health (NIH) as the collective genomes of the microbes (composed of bacteria, bacteriophage,

fungi, protozoa and viruses) that live inside and on the human body (Yang, 2012). They impact

our physiology, protect against pathogens, support immune system growth and development, and

have metabolic functions. There are nearly 100 trillion intestinal bacteria living in a human host

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and around 1000 different species found in the gut that are essential for health. In this paper, the

gut microbiome is referred to as the gut flora, gut microbiota, intestinal flora, or intestinal

microbiota. The symbiotic relationship between human host and intestinal microbiota provides

the gut flora with a nutrient-rich environment and the human host with nutrient metabolism and

immune support (Yang, 2012; Shreiner et al., 2015).

Gut microbes are essential for health and disease prevention. Gut microbial eubiosis (a

healthy balance of the microflora in the gastrointestinal tract) is essential for the human host and

gut microbiome to live in harmony and maintain optimum health and immunity. Infancy is

viewed as the critical window for the establishment of a healthy symbiotic relationship and

proper development of the intestinal barrier that affects intestinal homeostasis maintenance later

in life (Shreiner et al., 2015). During the start of gut microbiome research, it was initially

discovered that the gut flora are important for nutrient metabolism and the development of the

intestinal barrier, but further research showed a surplus of other beneficial function of the gut

microbiome. It was seen to inhibit pathogens, aid in the development and regulation of the

immune system, modulate gene expression, and play a major role in the gut-brain axis. This

paper focuses on its role in the development and regulation of an infants intestinal barrier and

immune system, and how formula feeding can disrupt the gut flora and negatively affect these

mechanisms, leading to increased risk of T2DM (Schreiner et al., 2015; Wang and Kasper, 2014;

Yoon et al., 2014).

Microbiota can be classified into two groups: Gram-negative and Gram-positive.

Microbial eubiosis is characterized by a positive ratio of gram-positive to gram-negative, and the

opposite is seen in microbial dysbiosis, which is an imbalance of gut microbiota with an

overgrowth of Gram-negative bacteria. Gram-positive bacteria are the predominant microbiota

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found in the small intestine that initiate proper development and utility of the gut, including

Bifidobacterium, Bacteroides, and Lactobacillus. One of the major benefits of Gram-positive

bacteria is their anti-inflammatory potential. Gram-negative bacteria, known to have a greater

prevalence in gut microbial dysbiosis and contribute to increased disease risk, include

Escherichia coli (E. coli), Pseudomonas aeruginosa, and Clostridium difficile.

Metabolites produced from gut bacterial metabolism are believed to provide many health

benefits to the human host and include lipids, bile acids, and short chain fatty acid (SCFA).

These metabolites contribute to pathogenic protection, immune homeostasis, metabolic pathway

regulation, and integrity of the epithelial barrier along with providing energy to the host cells.

The point of interest in this paper are SCFA and their ability to regulate immune function,

intestinal permeability, inflammatory responses, insulin secretion, and glucose metabolism. Such

SCFA include butyrate, acetate, and propionate (Sharon et al., 2014; Morrison and Preston,

2016).

Techniques to study microbiota diversity

Genes affect the development of the microbiome and vice-versa. A microbial imbalance

can negatively affect gene expression, and gene expression can affect the presence and function

of gut microbial species. Research on the metagenome of the intestinal bacteria environment has

dramatically increased within the past five years. The metagenome is the collection of all the

genetic material of individual organisms in a given environment. Metagenomics is the study of

the metagenome and is used to identify microbial diversity and function (National Research

Council). Metagenomic sequencing refers to culture-independent methods that focus on either

the structure or function of aggregate genomes in a community (Thomas et al., 2012).

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 The microbiome is individually tailored and varies from person to person, making it

somewhat difficult to study and compare microbial species and their effects. Techniques used in

research to identify and categorize individuals gut microbiota are culture based analysis, gene

fingerprinting methods (i.e. 16S rRNA gene pyrosequencing, qPCR amplification, random

amplified polymorphic DNA), Fluorescent In Situ Hybridization (FISH) analysis, and

metagenome shotgun sequencing. All of these methods, other than the cultured based analysis

used solely to isolate and identify species types, are used to establish meaningful gene profiles.

(Harmsen et al., 2000; Ahn et al., 2011; Weinstock, 2012).

Immune-Microbiome Co-Maturation
This paper discusses the microbiomes role in the development of the mucosal immune

system through intestinal development, intestinal-mucosal barrier development, and later-life

dietary metabolism. It also focuses on how mechanisms influence glucose homeostasis,

microbial translocation and inflammation in the development of T2DM. The intestinal-mucosal

immune system involves the bodys immune responses that occur in the intestinal mucosa of the

gastrointestinal tract (GI). The lymphoid tissue that makes up the GI tract is referred to as the

gut-associated lymphoid tissue (GALT). The initial development of GALT starts during the

narrow critical window in infants during which the development of the intestinal tract determines

the progression of the adult-like intestinal immune system, and optimal maturation depends

heavily on the presence of the gut microbiome (Round and Mazmanian, 2014).

The newborns sterile body is seeded with microbes from the mother, as well as early-life

nutrition and other environmental sources, immediately after birth and for months thereafter. The

gut, along with mucosal tissues and the skin, are established with specific microbial species that

support critical health-related activities: 1) postnatal development of the intestinal-mucosal

immune system, 2) immune-tissue homeostasis, 3) infant and later-life dietary metabolism, and

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4) restricted access of pathogenic gut microbes to environmentally-accessible sites, referred to as

microbial translocation (Dietert, 2013). The documented effects of the gut microbiota on

intestinal-immune homeostasis are shown in Figures 3 and 4 below (Lin and Zhang, 2016).

Figure 3: Effects of gut bacteria on intestinal-immune homeostasis. *SFB: Segmented

Filamentous Bacteria that induces postnatal GALT maturation and T cell compartmentalization.
IE: intestinal epithelium (Lin and Zhang, 2016).

Figure 4: Development and maturation of the intestinal mucosal barrier

and immune system CRAMP, cathelin-related antimicrobial peptide; CRS,
cryptidin-related sequence; TLR, Toll-like receptor; : cells might or might not be
present; +, ++, +++, ++++: relative numbers of indicated cells. (Jain and Allan,
2014)
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 Infant feeding and intestinal microbial colonization encourage maturation of the intestinal

mucosa (lining of the GI tract) and initiate development of the mucosal immune system.

Bidirectional interactions between gut microbiota, diet and the immune system itself regulate the

establishment and maintenance of intestinal homeostasis and barrier function. Neonatal immune

system maturation in mucosal tissues is greatly influenced by the microbiota with both innate

and adaptive immunity affected via the interactions in the symbiont (the human host). (Rakoff-

Nahoumand Medzhitov, 2006; Gaboriau-Routhiau et al., 2011; Chung et al., 2012). The gut

microbiome and mucosal immune system have a symbiotic relationship. The infants GALT

influences the individuals immune responses, glucose metabolism, and incidence of gut

permeability. Improper development of both the gut microbiome and GALT can lead to

complications involving metabolism, gut permeability, immune response, and inflammation

(Rakoff-Nahoumand Medzhitov, 2006; Gaboriau-Routhiau et al., 2011; Chung et al., 2012).

The intestinal mucosa consists of one or more outer layers of epithelial cells (enterocytes)

overlying connective tissue referred to as the lamina propria. The intestinal mucosa surrounds the

inner open space or cavity of the GI tract called the lumen, which is where the gut microbiota

reside (Figure 5). The non-adhesive enterocytes are held together by tight junctions, adhesive

junctions, and desmosomes. The tight junctions are important for regulating the barrier function

of the epithelium. They do so by facilitating the epithelial barriers selective permeability and

transport of substances between cells. Gut microbial composition and cytokines (i.e. tumour-

necrosis factor (TNF) and interferon- (IFN)) are known to regulate the permeability of the tight

junctions. Gut bacteria can promote or prevent production of cytokines that increase the

permeability of tight junctions. Gram-positive bacteria like Bacteroides, Bifidobacteria,

Streptococcus, and Lactobacillus have been seen to reverse the effects of cytokines (Resta-

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Lenert and Barrett, 2006). According to the findings of Jain and Allan (2014), the mechanisms

by which commensal microbiota protect barrier function incldue modulation of the expression of

the occludin and caludin protein and utilization of epithelial cell signalling machinery such as

Rho GTPases, PKC and MAPK pathways to enhance barrier integrity (Jain and Allan, 2014, pg.

7). Thus, the gut microbiome plays a major role in epithelial cell signaling pathways that control

intestinal permeability along with regulating gene expression of tight junction proteins (Resta-

Lenert and Barrett, 2006; Jain and Allan, 2014).

Mucosal immune system development begins in the fetal stage and is initiated by

lymphoid tissue under cells (LTi). Two of the first structures to form in the GI tract are Peyers

patches and mesenteric lymph nodes. The sites of developing intestines during this stage are

referred to as cryptopatches. The maturation of cryptopatches into isolated lymphoid follicles is

triggered by the activation of pattern-recognition receptors (PRRs) known as toll-like receptors

(TLRs) by microbe-associated molecular patterns (MAMPs). This maturation requires the

colonization of gut microbiota. Referring to Figure 5, the intestinal barrier is made up of a single

layer of epithelium consisting of: enterocytes (intestinal cells), specialized Goblet cells,

microfold (M) cells, Paneth cells (present only in small intestine), and enteroendocrine cells.

Dendritic cells are important for collecting and processing antigens from gut microbes and

delivering them to T-cells that initiate an immune response. Dendritic cells in the Peyer's patches

access microbial antigens through M cells or directly from the lumen by extending dendrites

through tight junctions. When the dendritic cells are full of microbial antigens, they induce T-cell

differentiation or T-cell-dependent B-cell maturation. T cells and B cells are lymphocytes

involved in immune response. T-cells can differentiate into T helper cells (TH), type 1 regulatory

cells (Tr1), or regulatory T cells (Treg). Figure 5 shows the intestinal barriers structure and

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composition involved in the maturation process of the intestinal barrier function (Jain and Allan,

2014).

Figure 5: The Intestinal barrier

Mulder et al. (2011) also explains the narrow critical window in infants during which the

seeding and development of the microbiome determines the progression of the adult-like

microbiome and intestinal maturation towards childhood and later on into adulthood (Mulder et

al, 2011). The individually tailored infant gut microbiome and the human host are symbiotically

joined. Any event that alters or inhibits this event will essentially produce an incomplete

human mammalian component, and this is probably the most significant sign that classifies the

individuals health and disease status. The importance of the completed self is described by

Dietert R and Dietert J (2012). A balance in the gut microbiome is shown in this paper to help

protect an individual from developing T2DM through its role in regulating the developing of

intestinal barrier and decreasing inflammation (Dietert and Dietert, 2012).

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Infant Formula and Diabetes Risk through the Gut Microbiome

The growth of the microbiome starts in utero and changes immediately post-delivery due

to exposure to bacteria from the mother, nurses and doctors, the hospital, and nutrition. It

continues to grow and change in number and species type. Seeding and nourishment is very

important for its development and proliferation (Dietert, 2013). Early life nutrition is one of the

major factors contributing to the initial growth and maturation of the gut microbiome. Breastfed

infants have been seen to have a different microbiome than formula-fed infants. This is shown in

multiple studies that this paper focuses on in a later section. Overall, breast fed infants have a

greater prevalence of gram-positive bacteria than formula fed infants (Dietert, 2013).

One particular gram-positive bacteria of interest in this paper is B. infantis. When B.

infantis digests HMOs, short-chain fatty acid (SCFA) metabolites are released from the

fermentation process that provide many benefits for the infants intestinal tract. Adenosine

triphosphate (ATP) and SCFAs produced by the metabolism of HMOs are utilized as energy

sources by intestinal cells. At birth, an infants intestinal tract is underdeveloped and highly

permeable due to gaps between gut cells. SCFAs are crucial for the maturation of the intestinal

tract by providing energy to gut cells and by modulating gene expression of tight junction

proteins that promote intestinal barrier integrity. The presence of B. infantis increases protein

expression of tight junction proteins including occludin and zonula occludens-1 (ZO-1) through

increased transcription of their genes. This suggests that gene expression, being modulated by

the gut bacteria, can regulate and improve intestinal barrier integrity (Ewaschuk et al, 2008).

Through these mechanisms, B. infantis and its metabolites encourage gut cells to make these

tight junction proteins that, in turn, seal the gaps found in the intestinal barrier during infant

development, decrease intestinal permeability, and keep microbes out of the bloodstream.

21
Compared to HMOs found specifically in breast milk, when B. infantis comes in contact with

lactose found in formula, it is unable to grow and flourish at the same rate or engage in the

promotion of gut cell gap closure of the intestinal barrier. (Ewaschuk et al, 2008; Scholtens,

2012).

By decreasing intestinal permeability, B. infantis essentially reduces the risk of bacterial

translocation of gram-negative bacteria into the blood. Gram-negative bacteria are known for

their pro-inflammatory effects in the body due to endotoxins found in their outer membrane (i.e.

lipopolysaccharide (LPS)). A greater prevalence of LPS in the lumen induces an inflammatory

response commonly seen in T2DM called metabolic endotoxemia (ME), which can be caused by

increased LPS levels in the blood from bacterial translocation or increased cytokine levels in the

blood produced by an innate immune response. The innate immune response is activated when

the body recognizes harmful antigens produced by gut microbes, like the endotoxin LPS, and

induces a pro-inflammatory cascade (Neves, 2013). In the pro-inflammatory cascade stimulated

by the presence of the endotoxin LPS, there are specific pattern recognition receptors (PRRs)

that recognize the presence of the LPS endotoxin: LPS-binding proteins (LBP), cluster of

differentiation 14 (CD14), an accessory protein (MD-2), and toll-like receptor 4 (TLR4). These

PRRs are all involved in a pro-inflammatory cascade. First, an LBP-LPS trigger complex is

formed when LBP binds LBS in the lumen. The LPB-LPS trigger complex activates TLR4 and

its co-receptors MD-2 and CD14. TLR4 activation recruits adaptor molecules (MyD88 and

TRIF) that activate specific kinases, leading to the transcription of pro-inflammatory molecules

through nuclear factor-kappa B (NFkB) transcription. NFkB is a pro-inflammatory transcription

factor in intestinal epithelial cells. Activation of NFkB produces pro-inflammatory molecules that

enter the blood stream including tumor necrosis factor- (TNF-), interleukin-1- (IL1), and

22
interleukin-6 (IL6) (Neves, 2013). Scientific evidence indicates an increased prevalence of these

pro-inflammatory molecules in insulin resistant individuals. Cytokines in the blood not only

affect insulin resistance but can also lead to beta-cell apoptosis and beta-cell failure, potentially

leading to T2DM (Pradhan et al., 2001; Banerjee, 2012; Lee et al,. 2008; Cani et al., 2008).

Evidence suggests that early life gut microbiota tailored to the infants system, such as

Bifidobacterium longum subsp. infantis, can depress pro-inflammatory cytokine levels (i.e. TNF-

alpha) and dampen down the inflammatory response at crucial windows of mucosal tissue

development. Improperly controlled inflammation in early life appears to promote late-life health

problems (Dietert, 2013, pg. 3). This paper provides evidence to suggest that formula feeding

alters the protective function of the intestinal barrier by two mechanisms: 1) preventing complete

maturation of the intestinal barrier, 2) promoting gut microbial dysbiosis and an increased

prevalence of Gram-negative bacteria (Neves, 2013; Rodes et al., 2013). Gut microbes impact

local and systemic inflammation through pattern recognition receptors (PRRs) (Claes et al.,

2015; Jin and Flavell, 2013). Intestinal dysbiosis, referring to alterations in the composition and

abundance of the gut microbiota as compared to healthy individuals, is believed to account for

inflammatory and metabolic diseases (Spor et al., 2011). Dysbiosis is the decline of gram-

positive bacteria such as Lactobacillus, Bifidobacteria, and Bacteroides and an increase in gram-

negative bacteria, such as Escherichia coli (E. coli), Pseudomonas aeruginosa, and Clostridium

difficile (Rodes et al., 2013; Neves, 2013; Claes et al., 2015; Jin and Flavell, 2013; Spor et al.,

2011).

Diet and other environmental and host factors have a major effect on gut microbial

composition, with the main focus of this paper being infant nutrition. A balanced microbial

composition results in eubiosis, which helps regulate immune and inflammatory responses

23
through the prdocution of anti-inflammatory and immunomodulatory products including SCFA,

which help maintain homeostasis and decreased risk of disease. Dysbiosis, an imbalance of gut

microbiota, can lead to dysregulation of the immune system through lack of beneficial

microbiota products like SCFA and an increased prevalence of pro-inflammatory factors like

lipopolysaccharides (LPS), leaving the host with greater susceptible to inflammation. Dysbiosis

could occur through the consumption of formula feeding, as well as through changes induced by

factors modulating gene expression (Figure 6) (Maslowski & Mackay, 2011)

Figure 6: Diet, microbial composition and regulation of the immune

system. (Maslowski & Mackay, 2011)

Microbial dysbiosis has substantial effects on inflammation, glucose metabolism, and

insulin resistance (Cani et al., 2008). Metabolic endotoxemia (ME) is a low-grade elevation of

plasma lipopolysaccharide (LPS) typically seen in individuals with T2DM. Lipopolysaccharides

are large molecules consisting of a lipid and polysaccharide chain composed of O-antigen. They

can be found in the outer membrane of gram-negative bacteria. Alterations in, or

24
underdevelopment of, the intestinal barrier can lead to increased intestinal permeability, favoring

translocation of microbiome-derived lipopolysaccharide to the bloodstream. LPS serum

concentrations have been shown to have a two- to threefold increase in the onset of ME.

Intestinal epithelium acts as a continuous barrier to avoid LPS translocation. The intestinal

barrier is regulated by proteins called zonula occludens 1 (ZO-1) and occludin (Cani et al.,

2008). Gut bacteria are known to modulate gene expression of these intestinal barrier-tight

function proteins (Hooper and Gordon 2001). The intestinal tight junction integrity helps keep

the microbiota above the epithelial surface and within the intestinal mucus. When the epithelial

barriers integrity is compromised from microbial dysbiosis, ME can come into play. ME has

been shown to trigger toll-like receptor 4 (TLR4) mediated inflammatory activation, eliciting a

chronic low-grade pro-inflammatory status which may result in insulin resistance and glucose

intolerance as seen in T2D. TLRs are pattern-recognition receptors (PRRs) located in enterocytes

(absorptive cells in the small intestine) (Cani et al., 2008; Hooper and Gordon, 2001).

Figure 7: Gut Microbiome on Health and Disease

25
 A microbial balance has proven to play a crucial role in regulating immune responses and

protection against inflammation-driven disease like T2DM (Figure 7). Many diseases are

characterized by pro-inflammatory cytokines such as tumour-necrosis factor (TNF) and

interferon- (IFN). These cells are characterized by their expression of pro-inflammatory

cytokines. Specialized T cells, known as regulatory T (Treg) cells, help reverse pro-

inflammatory responses and maintain immune homeostasis. A study by Round and Mazmain

(2014) suggests that intestinal bacteria interact with the mammalian immune system to direct the

linage differentiation of both pro- and anti- inflammatory T-cell populations. This study suggests

that a population of inflammatory T cells, termed T helper 17 (TH17) cells, has been implicated in

the pathogenesis of T2DM. It is possible that different types of bacteria induce diverse

immunological functions, thus, the equilibrium between inflammation and regulation in the gut

may be due to the mutualistic community structure of the microbiota (Round and Mazmanian,

2014).

Microbiome as a Mediator for Association between BF and T2D

Breastfeeding & the Microbiome

In a review by Rodney R. Dietert from Cornell University (2013), recent evidence

suggests that BF can positively restore the gut microbiome and, in turn, improve immune

homeostasis and reduce the risk of chronic disease later in life. This review successfully links

microbial dysbiosis and breastfeeding to the pathogenesis of T2DM (Dietert, 2013). According

to research, Clostridia, Enterococci, and Enterbacteria spp. levels are increased during a

microbial dysbiosis occurrence. One study in particular by Harmsen et al. investigated the

development of the gut flora in breastfed and formula-fed infants using new molecular

identification and detection methods. Previous studies have shown that assessing the infant gut

26
flora from fecal samples lacked accuracy with cultivation methods due to nonspecific media and

bacterial species that cannot be cultured. The goal was to provide more precise data on the

diversity and progression of bacterial strains within 20 days after initial neonatal gut colonization

(Dietert, 2013; Harmsen et al, 2000).

This prospective cohort study looked at the development of the infant gut microbiome in

a group of 12 infants, 6 breast-fed and 6 formula-fed, during the first 20 days of life (Harmsen et

al, 2000). The bacterial population and dominant species were compared between breastfed and

formula fed infants. All of the infants were either born at home or in the hospital in which they

were there for no longer than 2 days. Six of the mothers exclusively breastfeed for the first 20

days of life and the other six intended to bottle-feed their infants for the first 20 days of life. The

mothers who intended to feed their infants formula were given a standardized whey-predominant

infant formula in order to prevent variance in data among the formula-fed group (Harmsen et al.,

2000).

The mothers collected their infants fecal matter throughout the study that was analyzed

using three different methods to identify the type and quantity of colonies. These methods were

culture-based analysis, amplified polymorphic DNA (RAPD) fingerprinting, 16S rRNA

sequence, and Fluorescent In Situ Hybridization (FISH) analysis. The RAPD fingerprinting and

16S rRNA sequence analysis (PCR sequencing) were used to group and identify microbial

species based on their patterns. For the FISH analysis, the total number of cells was counted

from each sample (Harmsen et al, 2000).

The data collected from the culturing on selective media and RAPD-PCR fingerprinting

showed that the main flora of both the breastfed and formula-fed infants consisted of 10 to 20

different RAPD groups. The dominant species called Bifidobacterium spp. grew on most of the

27
selective media, and unwanted species also grew on some of the plates. Bifidobaceria and

Escherichia coli were the dominant normal flora in both groups, along with Enterococci,

Staphylococci, Bacteroides, and Veillonella. The culture-based analysis showed that lactic acid

bacteria and Bifidobacteria had a greater prevalence in the breast-fed infants, whereas the

formula-fed infants were more established with Bacteroides and Clostridia spp. The FISH

analysis showed that Bifidobacteria dominated the breastfed infants gut between days 12 and 20

(60-91% of the flora). In the majority of formula-fed infants, the Bacteroides population

increased toward day 20 and E. coli was also more prevalent (Harmsen et al, 2000).

The results confirmed that Bifidobacteria became the dominant bacteria (>60%) in

breastfed infants within one week after birth. Along with Bifidobacteria being the dominant

bacteria in breastfed infants, they also had higher numbers of lactic acid bacteria like

Streptococci and Lactobacilli. Formula-fed infants had more staphylococci and clostridia. This

could explain the low pH buffering capacity of breast-milk. The goal of this study is to help in

the development of an infant formula that will induce breast milk-like colonization in the infant

gut. It is known that Bifidobacteria and lactic-acid producing bacteria aid in the maturation of the

mucosal immune system and have anti-inflammatory effects (Harmsen et al, 2000). The study by

Harmsen et al and various others have shown similar results in the gut microbiome of breast fed

and formula fed infants (Table 1). In all these studies, Bifidobacteria became the dominant

bacteria in the infant fed breast milk, along with a majority of the studies showing increased

prevalence of Gram-positive bacteria like Actinobacteria, Lactobacillus, and Streptococcus,

Clostridium Perfringens, and Enterococcus. The results show that formula fed infants have

higher levels of Gram-negative bacteria in the gut, including Clostridium difficile, Escherichia

28
 coli, Akkermansia, Lachnospiraceae (associated with T2DM in a metagenome study (Qin et al,

2012)), Prevotella, and Bacteroides fragilis.

Table 1: Gut Microbial Composition in BF vs FF infants

Subjects Analyzed & Results (detected bacteria)

References Methods Breast fed Formula fed
Harmsen et al, n=12 infants (6 BF and (% of fecal matter): (% of fecal matter):
2000 6 FF) 60-91% Bifidobacterium 28-75% Bifidobacterium
11-50% Bacteroides 35-61% Bacteroides
Fecal samples collected
at 20 days *only found in BF infants: *only found in FF infants:
Lactobacillus Staphylococcus aureus
Streptococcus Clostridium ramnosum
Clostridium Perfringens Brevibacterium iodinum
Azad et al, 2013 n=24 (10 exclusively Dominant bacteria: Dominant bacteria:
BF, 5 partially BF Bifidobacterium Clostridium difficile
(supplemented with Firmicutes Akkermansia
formula), 9 FF only) Streptococcus Lachnospiraceae

Fecal samples collected Overall: increased richness and

at 3-4 months higher diversity

Liu et al, 2015 Human study: n=120 Human study:
(7-90 days old: 40 BF, Bifidobacterium (8.17 0.3)
40 fed SPCF (Synlait Lactobacillus (6.90 0.20)
control formula), 40 Clostridium perfringens (4.80
FF); Fecal samples 0.20)
collected on day 1 and
day 21 Rat study:
Clostridiales (14.1 1.34)
Porphyromonadaceae (13.8
Rat study: n=12 (6 fed 1.94)
human milk, 6 fed Collinsella (7.04 1.71)
SPCF); rats fed for 28 Prevotella (4.76 2.32)
days Mucispirillum (0.03 0.02)
Enterobacteriaceae (0.08
0.06)
Solis et al, 2010 n=20 (20 exclusively (% of fecal matter)
BF infants)
Day 10:
Fecal samples collected Bifidobacterium (60%)
at 1, 10, 30 and 90 days Streptococcus (13%)
of age Lactobacillus (8%)
Enterococcus (8%)
Human study:
Bifidobacterium (6.94 0.3)
Lactobacillus (6.65 0.25)
Clostridium perfringens (4.65
0.21)
Rat study:
Clostridiales (5.10 1.20)
Porphyromonadaceae (7.69
2.00)
Collinsella (2.76 0.05)
Prevotella (29.7 8.61)
Mucispirillum (not detected)
Enterobacteriaceae (not
detected)
Not included

Day 90:
Bifidobacterium (42%)
Streptococcus (26%
Lactobacillus (16%)
Enterococcus (16%)
Schwartz et al, n=12 (6 exclusively BF Higher levels of: Lower levels of:
2012 and 6 FF) Bacteroidetes Actinobacteria

29
 Proteobacteria Proteobacteria
Fecal samples collected Actinobacteria
at 3 months of age Higher levels of:
Firmicutes

Not detected:
Bacteroidetes
Penders et al, 2006 n=1030 (700 Dominant bacteria: Dominant bacteria:
exclusively BF, 232 Bifidobacterium Bifidobacterium
exclusively FF, 98 BF
and FF) Higher levels of:
Escherichia coli
Fecal samples collected Clostridium difficile
at 1 month of age Bacteroides fragilis
Bezirtzoglou et al, n=12 ( 6 BF, 6 FF) Dominant bacteria: Dominant bacteria:
2006 Bifidobacteirum (69.12%) Bifidobacterium (32.07%
Fecal samples collected
during first 2 months of Followed by: Followed by:
life Bacteroides (11.85%) Bacteroides (28.73%)
Prevotella Atopobium (6.82%)
Staphylococcus E. coli
Lactobacillus Clostridium
Enterococcus Streptococcus
Streptococcus Staphylococcus

Gut Microbiome and Type 2 Diabetes

Early signs of an increased risk for insulin resistance and T2DM can be detected by

looking at an individuals microbiome (Burcelin et al, 2011; Lee CC et al, 2008). Evidence

suggests that a dysbiosis of the gut microbiome may potentially lead to development of T2DM

due to the occurrence of different mechanisms that occur simultaneously. These events include:

1) TLR mediated inflammatory cascade resulting in increased cytokine production in response to

high numbers of Gram-negative bacteria in the lumen, 2) the passage of pro-inflammatory gut

bacteria into the underlying intestinal tissues and into the bloodstream, 3) an innate inflammatory

response to increased serum levels of inflammatory markers, 4) increased cytokine levels that

contribute to poor glucose homeostasis, decreased insulin secretion and insulin resistance, thus,

resulting in the onset of T2DM.

30
 Groundbreaking research demonstrates the association of T2DM with the presence of

Gram-negative gut bacteria and the bodys innate immune response. Individuals with T2DM

have been shown to have higher levels of Gram-negative bacteria in the gut that are contributing

to the presence of gut microbial dysbiosis. A study by Larsen et al. (2010) examined the fecal

bacterial composition of 36 male adults, with 18 subjects diagnosed with T2DM. Compared to

the control group (individuals without T2DM), the diabetic group had significantly reduced

levels of Firmicutes with increased levels of Prevotella and Bacteroidetes. Higher levels of

Prevotella and Bacteroidetes were positively correlated with higher plasma glucose levels and

reduced glucose tolerance. This study provides evidence showing that gut microbial composition

in diabetic patients differs to that of non-diabetic individuals, suggesting that the microbiota

plays a role in the onset of the disease.

A metagenome-wide association study by Qin et al. (2012) provides evidence showing

patients with type 2 diabetes were characterized by gut microbial dysbiosis. Along with

identifying the presence of gut microbial dysbiosis, the study also identified 60,000 T2DM-

associated metagenomic markers. Qin et al. carried out their analysis through deep shotgun

sequencing of the gut microbial DNA of 345 Chinese individuals. The results revealed that the

diabetic patients had higher levels of Gram-negative microbes in their gut, and less microbes

involved with SCFA production. They found that the increased prevalence of Gram-negative

bacteria involved in mechanisms that lead to T2DM. When analyzing their data, metagenomic

linkage groups (MLG) were devised in order to reduce the large amount of data collected and

because the metagenome works as a functional unit rather than independently. 84.4% of the

MLGs that they identified were T2DM-associated gene markers, and most of them were linked

to opportunistic pathogens like Escherichia coli, Bacteroides caccae, and Clostridium ramosum.

31
These bacteria are known to cause ME. The gut microbiota that were seen in the T2DM patients

were associated with poor glucose homeostasis, sulphate reduction, oxidative stress and

decreased butyrate production. Despite the fact that the degree of gut microbiota dysbiosis was

less significant than that seen in inflammatory bowel disease, the markers found in the T2DM

still play a role in the development of T2DM and the inflammation that characterizes the disease

(Qin et al, 2012).

The functional links between gut microbiota, inflammation, and metabolic diseases have

recently been illustrated by multiple studies. Many of the studies explain the reason for increased

levels of the positive acute-phase protein CRP seen in T2DM patients. CRP is seen to increase in

response to a rise in the major cytokines like tumor necrosis factor- (TNF-), interleukin-6 (IL-

6), interleukin 4 (IL-4), and interleukin 1 (IL-1). These cytokines are derived from adipose tissue

and are referred to as adipokines, which can explain the link between obesity and T2DM. In a

study by Bindels LB et al, restoration of Lactobacillus spp. correlated with a drop of

inflammatory cytokines including IL-6, IL-4, and granulocyte colony-stimulating factor

(Bindels et al, 2012). In T2DM, these cytokines, along with cytokines produced by an innate

immune response discussed later, directly affect insulin resistance as well as beta-cell function.

Low-grade inflammation seen in individuals with T2DM may explain the reason for insulin

resistance and declined pancreatic insulin secretion that characterize the disease. Cytokines

directly inhibit insulin receptor signaling and leads to phosphorylation of insulin receptor

substrate-1. The inhibition of insulin receptor signaling by TNF- and IL-6 promotes hepatic

fatty acid syntheses, a process that is normally suppressed by the binding of insulin to receptors

on adipocytes (fat cells). Increased hepatic fatty acid synthesis induces the liver to recruit more

inflammatory markers to adipose tissue and pancreatic beta-cells as well as produce more acute-

32
phase proteins, mainly CRP. Cytokines not only affect insulin resistance but can also lead to

beta-cell apoptosis and beta-cell failure, leading to T2D (Lee et al, 2008).

Microbial dysbioses is perceived as a risk for T2D by promoting abnormal energy

metabolism along with inflammation caused by intestinal permeability (IP) and increased

metabolic endotoxin secretion (Cani et al, 2008; Diamant et al, 2011; Everard, 2013). Increased

metabolic endotoxin secretion results in metabolic endotoxemia, which has been shown to

initiate insulin resistance and other factors related to T2DM. IP was seen to be affected by the

gut microbiota, and in turn affect the levels of circulating inflammatory markers in a study by

Leclercq et al (2014). This study tested the correlations between gut bacteria and IP. Data

analysis revealed a positive correlation between IP and Lachnospiraceae dorea. Bacteria

belonging to Ruminococcaceae and Bifidobacterium families were negatively coorelated with IP.

These results support the hypothesis that the increased Gram-negative bacteria in the lumen

influences gut-barrier function. (Leclercq et al, 2014).

In a 4 week clinical trial on mice, Cani et al (2008) investigated the role of the microflora

on metabolic endotoxemia, intestinal permeability, adipose tissue inflammation, glucose

intolerance, and insulin resistance. Cani et al. found that an increased gram negative-to-gram

positive ratio leads to increased plasma levels of LPS. A change in gut bacteria results in

increased intestinal permeability and LPS levels in the blood that are responsible for metabolic

endotoxemia, adipose tissue inflammation, increased blood glucose profiles, insulin resistance,

and reduced insulin secretion. Increased gut permeability was seen to enhance LPS levels from

bacteria crossing the intestinal barrier, all due to gut dysbiosis characterized by an increased ratio

of gram negative-to-gram positive microbiota (Cani et al, 2008).

33
 These dysbiosis findings were demonstrated by a 4-week long antibiotic treatment in a

total of 38 laboratory mice. These mice were divided into three groups: controlled diet and

antibiotic treatment (n= 13), high-fat diet and antibiotic treatment (n= 17), and ob/ob mice only

treated by antibiotics (n=8). The clinical trial aimed at changing the gut microbiota and providing

mechanisms responsible for its effect on the occurrence of metabolic endotoxemia and the onset

of corresponding metabolic disease such as T2D. Previous studies have suggested the link

between gut microbiota and endotoxemia and endotoxemia and metabolic disease, but this study

is the first to show the link between all three. Bifidobacteria spp. and Lactobacillus spp. were

detected from the collected cecal contents by qPCR amplification and 16S rRNA gene

sequencing. When looking at intestinal permeability, gut microbial dysbiosis reduced the

expression of epithelial tight junction proteins such as ZO-1 and occludin, resulting in increased

intestinal permeability and bacterial translocation (Cani et al, 2008).

Results from this study by Cani et al. (2008) prove that a change in gut microbiota can

improve glucose tolerance. Glucose-induced insulin secretion, insulin resistance index, body

weight gain, and adipose weight were all corrected by the change in gut microbiota, favoring a

decreased gram negative-to-gram positive ratio. Metabolic endotoxemia and inflammation were

also reduced in all mice treated with antibiotics, regardless of feeding type. When looking at

adipose tissue inflammation in antibiotic treated mice, the adipose depots contained lower levels

of inflammatory markers of metabolic disorders, including PAI-1, IL-1, TNF-, and F4/80.

Inflammation in T2D is often associated with oxidative stress in adipose tissue, leading to

increased mRNA concentrations of these inflammatory markers. A change in microbiota toward

a decreased gram negative-to-gram positive ratio led to a reduced occurrence in these

34
inflammatory markers, resulting in reduced manifestations of intestinal permeability, metabolic

endotoxemia, blood glucose profiles, and insulin resistance (Cani et al, 2008).

Peroxisome proliferator-activated receptor (PPAR) is a well-recognized transcription

factor to link metabolism to inflammation in the intestine. In addition to the GI tract, PPAR is

predominantly expressed in adipose tissue and thus participating in the metabolic regulation of

lipids, glucose homeostasis, cell proliferation and differentiation as well as local inflammation

(He et al, 2015; Saponaro et al, 2015). Streptococcus salivarius had previously been shown to

influence inflammation by down-regulating NF-B in the intestinal cells (Kaci et al, 2014), and

has recently been revealed to inhibit transcriptional activity of PPAR (Couvigny et al, 2015).

Upon exposure to S. salivarius supernatant, the expression levels of I-FABP (intestinal fatty acid

binding protein) and Angptl 4 were found to markedly drop among PPAR-induced metabolic

genes in the IECs (Couvigny et al, 2015). Altogether, studies strongly suggest that gram-negative

gut bacteria could exert dual effects on both host inflammatory regulation and metabolism

processes. (Kaci et al, 2014; Couvigny et al, 2015; He et al, 2015; Saponaro et al, 2015).

Breastfeeding and Type 2 Diabetes

Infant Feeding, Insulin Signaling & Glucose Homeostasis

Formula feeding or short duration breastfeeding that has been seen to produce an

increased gram negative-to-gram positive ratio of gut microbiota can potentially cause metabolic

endotoxemia and eventually lead to the onset of T2D. FF infants are found to have lower levels

of anti-inflammatory microbial species residing in the lumen. Supportive results of exclusive

breastfeeding and regulated cytokine production were found in a study by Belderbos et al.

(2012). This human prospective birth cohort study of 291 healthy term infants compared

35
breastfeeding to formula feeding and their ability to modulate innate immune function.

According to Belderbos et al. (2012), breastfeeding was the major determinant of neonatal

innate immunity, associated with 5 (31%) of neonatal innate immune parameters, of which the

association with TLR7-mediated IL-10 production was most significant (76 pg/ml in breastfed

neonates vs. 293 pg/ml in formula-fed neonates, p = 0.001) (Belderbos et al., 2012, pg. 65). The

results show that exclusive breastfeeding during the first month of life was associated with

decreased TLR mediated cytokine production (twofold decreased TNF- and fourfold decreased

IL-10) compared to formula fed infants. When testing the concentrations of T cells, there was no

association between breastfeeding and concentrations or a difference between BF infants and FF

infants (Belderbos et al, 2012).

A study by Mayer-Davis et al. (2008) looked at the effects of breastfeeding on T2DM.

The 3 year long multicenter retrospective cohort study included 247 participants under the age of

21 from different race/ethnic groups (African-American, Hispanic, and non-Hispanic white).

History of BF was obtained from the mother. Information on duration and timing of solid food

introduction was obtained. 80 participants had T2DM and 167 were non-diabetic. Results show

that 31.3% of youth with T2DM were BF, whereas 63.5% of non-diabetic control youth were

BF. Duration of BF was also longer in the non-diabetic control group (P < 0.0001). T2DM case

subjects had lower odds of being BF in infancy. There was no difference observed according to

race/ethnicity. Overall, there were higher proportions of breastfeeding in the non-diabetic control

group. These findings suggest the BF elicits protective benefits against the development of

T2DM (Figure 8) (Mayer-Davis, 2008).

36
 Figure 8: Percent of Youth Ever Breastfed By Race/Ethnicity. Black column:
T2DM. White column: non-diabetic control

In a study published in 2011 by Veena et al., prolonged breastfeeding was associated with

protection against glucose intolerance at 9.5 years, suggesting that exclusivity and duration can

modify the extent to which breastfeeding protects against disease. This prospective cohort study

followed Indian children from ages of 1 to 9.5. Researchers assessed breastfeeding duration and

age at introduction of solid foods of 568 children at 1, 2 and 3 years of age. 518 children were

assessed for glucose intolerance and insulin resistance was at 5 and 9.5 years of age. Results

show that all children were initially BF, 90% were exclusively BF for at least 6 months, and 64%

were BF for 12 months or longer. At the 5 year mark, increased duration of BF was associated

with decreased insulin resistance and increased glucose concentration. At 9.5 years, glucose

concentration decreased and insulin resistance was unrelated to BF duration. The results show

that longer BF duration is associated with lower fasting insulin concentrations and insulin

resistance at 5 years, but not 9.5 years (Veena et al., 2011).

37
Infant feeding, Gut Microbiome & T2D

Diehl et al. (2013) revealed in a mouse model that microbial eubiosis in BF infants helps

restrict the transport of commensal and pathogenic bacteria in the gut to the mesenteric lymph

nodes. The mesenteric lymph nodes are sites of immune response stimulation and help regulate

inflammatory response. This microbial-immune homeostatic regulation limits microbial

translocation and the likelihood of misregulated inflammation, which could help to prevent

T2DM (Diehl et al, 2013). Early life nutrition through breast-milk or formula milk and solid

weaning foods is a key determinant of early microbial community structure that influences

development of protective immune responses and affects long-term health status and risk of

disease. Diet-induced microbial dysbiosis can lead to immune-mediated inflammatory and

metabolic diseases like T2DM. The mucosal barrier development, influenced by infant feeding,

carries with it innate and adaptive defenses and intestinal barrier function. The infant feeding,

microbiota and immune system trio strongly influences postnatal intestinal homeostasis, which

has consequences into adulthood. Figure 9 depicts this triad and the maintenance of intestinal

and metabolic homeostasis (Jain and Allan, 2014).

Figure 9: Diet, gut microbiota and host immunity (Jain and Allan, 2014)

38
 Dietert R confirms that breastfeeding is one of the longest standing and most effective

measures to protect against the development of childhood and adult disease. Dietert R focused on

the reduced risk of inflammation-driven chronic disease (i.e. T2DM) via immune-microbiome

co-maturation as a result of prolonged infant breastfeeding. Prolonged breastfeeding, defined as

at least 4-6 months of exclusive breastfeeding, along with the effects of natural birth starts the

initial seeding of the microbiome and contributes beneficial factors to its growth and maturation

including maternal antibodies and immune cells, additional bolus of microbes, tolerating

cytokines and antigens, and oligosaccharides tailored to specific microbes (Figure 5). These

factors and nutrients provide the necessary components to the completed self and help

diminish immune dysfunction and misregulated inflammation as seen in T2DM (Dietert, 2013).

Recent research suggests that breastfeeding provides the necessary components for both

the completion of the human superorganism self-completion (symbiotic relationship between

the human host and gut microbiome) and effective gut-bacterial supported immune maturation

and homeostasis. Immune maturation and homeostasis is needed to minimize immune

dysfunction, misregulated inflammation, and an elevated risk of T2DM (Figure 10) (Dietert,

2013). Other studies also indicate the significance of the susceptibility of metabolic processes

related to T2DM related to infant feeding through gut metagenomic identification. A

codependent relationship is observed between microbiota and human host genes. This

relationship has a significant effect on immune system related activities including inflammatory

response, transport, and metabolism. A transition toward studying the link between infant

feeding, microbiota and host genes on a system level approach rather than on an individual level

has resulted in stronger evidence regarding the relationship between microbial community and

host immune system. Mapping the interactions between type of infant feeding, human genes, and

39
gut microbiota colonization was a major advancement achieved in a recent study (Praveen et al.,

2015).

Figure 10: Combined Effects of Natural Birth and Breastfeeding

on Immune-Microbiome Development.

In a study by Praveen et al. (2015), the reanalyzed metagenomic data came from

Schwartz et al. (2012) and the reanalyzed transcriptomic data was obtained from Chapkin et al.

(2010). Praveen et al. aimed at using both data collections to provide evidence for the relation

between the microbiota, the human host system, and infant feeding mode. In the study by

Chapkin et al., a novel molecular methodology was developed that quantified intestinal gene

expression profiles in the developing infant by utilizing stool samples containing intact sloughed

epithelial cells. To identify gene sets that control intestinal development and function, they

collected fecal samples from 12 exclusively BF infants and 10 formula fed infants at 3 months of

age. They found that gene expression of specific proteins that are important for the development

and function of the GI tract was differentially regulated in response to infant feeding (Praveen et

al., 2015; Chapkin et al, 2010).

40
 In the Chapkin et al. (2010) study, genes expressed at higher levels in the BF infants were

EPAS1 (3.3 ratio), NR3C1 (5.513 ratio), and NR5A2 (2.837). The EPAS1 gene codes for an

important regulatory protein hypoxia-inducible factor (HIF). HIFs have been seen to affect

glucose metabolism, intestinal permeability, lipid peroxidation, and interleukin 1 beta (IL-1)

gene expression (Kannan et al, 2011). The NR3C1 gene codes for glucocorticoid receptors

(GCR) that are known to regulate the bodys inflammatory response. GCRs modulate genes that

control the development of the GI tract, metabolism, and immune response. This anti-

inflammatory receptor increases expression of other anti-inflammatory genes like interleukin-10

(IL10) and inhibits expression of pro-inflammatory genes like interleukin-1 (IL1). GCR also

regulates the activation of NFkB (the transcription involved in the TLR4-mediated inflammatory

cascade in response to endotoxin LPS). It suppresses NFkB by binding to its co-activators and

acting as a receptor antagonist by inhibiting the binding of pro-inflammatory molecules. Thus,

GCR helps restrict a rise of cytokines in the blood (Chapkin et al., 2010; Kagoshina et al., 2003).

The NR5A2 gene codes for a protein that induces cell protection and proliferation

referred to as liver receptor homolog-1 (LRH-1). LRH-1 protects pancreatic islets and -cells

from cytokine-induced apoptosis, resulting in decreased impaired glucose-induced insulin

secretion caused by increased cytokine levels (Baquie et al., 2011). Overall, these genes help

regulate glucose metabolism, intestinal permeability, inflammation, and insulin secretion. An

increased prevalence of protective genes like EPAS1, NR3C1, and Nr5A2 is one way BF plays a

major role in protecting the infant against future development of T2DM. In the Schwartz et al.

study, the microbiome was shown to be a mediator between infant feeding mode, intestinal

development and function, and gene expression (Baquie et al., 2011; Chapkin et al., 2010)

41
 The study by Schwartz et al. (2012) examined the correlation between host intestinal

mRNA gene expression and genes in the gut metagenome of exclusively breast-fed (BF) and

formula-fed (FF) infants at 3 months of age. They tested the hypothesis that this correlation

influences important regulatory pathways of the microbiome affecting intestinal development in

the first few months of life. Six mothers of BF infants and six mothers of FF infants participated

in the study. Stool samples were collected and researchers extracted and sequenced microbial

DNA. The mRNA from host gut exfoliated epithelial cells was also isolated from the stool

samples and were processed for microarray analysis. After these two operations were conducted,

the data was analyzed and correlations between the gut microbial metagenome and epithelium

transcriptome (the set of all mRNA molecules in a population of intestinal epithelial cells, in this

case) were identified. They demonstrated the relationship between gut microbiota genes and the

pathogenesis of bacterial colonization and the impact that these genes have on the host

metabolism and immune mechanisms. The gut metagenome in this study differed between BF

and FF infants. This difference signifies a beneficial effect of human milk and its factors on the

mutual relationship of the gut microbiome, genes, and maintaining intestinal homeostasis.

(Schwartz et al. 2012).

The type of feeding in this study influenced the microbe-gene co-expression network. In

the study by Praveen et al., data from Schwartz et al. was looked at in a biological system

approach. The results indicate that the type of infant feeding affects the expression of genes

along with the microbial community, and that these two factors influence the host immune

system. Breastfed infants had a microbial-gene network with a greater number of interactions

(~2.1 times the FF network), independent from the diversity of gut microbial species. The type of

infant feeding affects the microbial composition along with gene expression in epithelial cells.

42
Although FF infants in this study had greater microbial diversity, BF infants had a denser co-

expression network, which is important for regulating intestinal homeostasis and the numerous

pathways involving inflammatory response and glucose metabolism. Connectivity in the

microbial-gene network was measured, and the denser BF network presented more dependencies

among the genes shown by multiple paths and short path lengths concerning the reachability to

nodes (between genes and microbes) as seen in Figure 6a and Figure 6b (Schwartz et al. 2012).

Figure 6 A: Plot showing the distribution of shortest path lengths of a host gene-
microbe network under FF and BF conditions. B: The plot represents the number of
nodes that can be reached (Y-axis) after traversing through certain path lengths, (here
1, 2, and 3 along X-axis). The node type referred here are the DE genes
(differentially expressed genes) from gene expression data, Species are the
microbes, and Transient nodes are the genes mined from literature that were found
to share relationship with microbial species (Schwartz et al. 2012).

Results from this metagenomic study (analyzed in two individual research articles)

provide evidence for the relationship between breastfeeding and decreased risk of developing

T2DM mediated by the gut microbiome. Immunity and mucosal defense-related genes reacted in

response to microbial conditions. Genes that regulate immunity and mucosal defenses along with

43
gut motility and epithelial homeostasis were seen in higher levels in BF infants compared to FF

infants. A synergistic relationship was present among specific immunity/defense-related genes

that were identified in the study (Schwartz et al. 2012).

Summary and Future Research Needs

This paper suggests the association between the gut microbiome, infant feeding, and type

2 diabetes risk. Major triggers of inflammation due to the imbalance of the gut microbiome are

shown to increase the risk of developing T2DM. The gut microbiome is a crucial contributor to

immune function and controlling gut permeability and metabolic endotoxemia. It is possible that

the microbiome plays a major role in the pathogenesis of T2DM through specific processes that

lead to chronic low-grade inflammation. An altered gut microbiome seen in FF infants can

potentially lead to improper development of the intestinal barrier and diminish intestinal

homeostasis. There is sufficient evident to suggest that formula feeding can reduce the integrity

of intestinal-immune homeostasis, thus leading to increased gut permeability (Cani et al, 2008;

Chapkin et al., 2010), translocation of pro-inflammatory commensal bacteria (Cani et al., 2008;

Hooper and Gordon, 2001; Diehl et al., 2013), and low-grade inflammation in the body (Neves et

al, 2013; Leclercq et al, 2014; Qin et al, 2012; Cani et al, 2008). An increase in inflammatory

proteins during the inflammatory state seen in T2DM is seen to alter insulin signaling,

potentially leading to reduced glucose-induced insulin response and eventually insulin resistance

in T2DM. Due to this series of mechanisms, FF infants who tend to have increased levels of pro-

inflammatory gut microflora may lack proper maintenance of intestinal homeostasis, resulting in

an increased risk for developing insulin resistance compared to BF infants.

Not only does the hypothesis that breastfeeding promotes a balanced gut microbiome

which, in turn, protects against T2DM make sense physiologically, but sufficient evidence seems

44
to support this claim. More than one mechanism was observed in the development of T2DM

caused by a dysbiosis of the gut microbiome seen in FF infants. The main mediator was the

inflammatory process and how its affected by an altered microbiome and poor intestinal-

immune homeostasis. Inflammation is potentially caused by improper development of the

intestinal barrier during infancy. This is due to decreased production of tight junction proteins

which can lead to increased intestinal permeability, an increased prevalence of Gram-negative

bacteria in the lumen which can spark a TLR response and lead to increased cytokine production

by activated NFkB transcription, increased innate inflammatory response to the presence of

Gram-negative bacteria in the blood from bacterial translocation, and the potential for metabolic

endotoxemia to destroy tissues and lead to poor glucose homeostasis, decreased insulin secretion,

and insulin resistance.

Discovering how breast milk influences a baby's gut bacteria could potentially help

scientists figure out the best way to feed premature babies, design better infant formulas, and

promote lifelong health. Little research has been conducted regarding how breast milks

components work in the body, how it affects the development of the gut microbiome and

intestinal tract, and the exact mechanism involving its protection against disease. Also, breast

milk composition varies between women, making it difficult to study and produce valid results.

The study by Harmsen et al. (2000) depicted the increased prevalence of Gram-positive

bacteria in the intestines of BF infants, like Bifidobacteria, Lactobacillus, and Streptococcus.

The study was limited, though, due to its small sample size and lack of inclusion criteria for the

mothers. This could potentially weaken the results and lead to bias. During the cultivation of

bacteria, Bacteroides were particularly difficult to culture, which led to different results based on

previous studies. Also, the plates had poor selectivity and were mostly dominated by

45
Bifidobacteria, Bacteroides, and E. coli. Despite these weaknesses, the results of this study were

similar to previous studies that compared the gut flora of newborn breastfed and formula-fed

infants. In all of them, Bifidobacteria was the dominant bacteria in the breast-fed infants, and this

bacteria is known to promote eubiosis.

Other studies that looked at the gut microbiome in BF and FF infants provide further

evidence for the presence of dysbiosis in FF infants, which can increase there risk of developing

T2DM. Azad et al. (2013) included infants that were born by cesarean delivery, mothers that

received antibiotics during pregnancy or at delivery, and infants that received antibiotics directly.

Cesarean deliveries and antibiotic use are also known to alter the gut microbiome, therefore the

results are completely valid when it comes to associating infant feeding with gut microbial

composition. There was also insufficient power for detecting differences of infant formula

brands in this study. Different brands of infant formula have different nutrition composition and

ingredients and can increase the variance of results amongst the FF infants. Penders et al. (2006)

also showed that cesarean section birth, hospitalization, prematurity, and antibiotic use affected

the prevalence of bacteria and were associated with decreased Bifidobacterium and Bacteroides

and higher levels of C. diff. It was seen that term infants who were born vaginally at home and

breastfed exclusively had greatest prevalence of beneficial gut bacteria, which suggests that age

of birth, type of delivery, and exclusivity and duration of BF all have affects on gut microbial

composition.

For studies that analyzed the presence of the gut microbiome by sampling feces, improper

feces collection by the parent can lead to inaccurate results. Questionnaires may not always be

accurate. The studies that had exclusion criteria involved with insufficient amount of feces, feces

collected at the wrong age, and incomplete questionnaires resulted in stronger evidence (Penders

46
et al., 2006). Also, results may be skewed if the breastfed infant wasnt exclusively breast fed,

and its hard to keep track of this in the study participants. Exclusivity and duration play major

roles in the type of bacteria that will show up in the results, as well as the brand of infant formula

use. When it comes to obtaining the full benefits of breastfeeding on the gut microbiome

development, it is recommended that infants are exclusively BF for at least 6 months. This can

increase levels of Bifidobacterium and decrease levels of C. diff and E coli.

Belderbos et al. (2012) successfully showed how exclusive breastfeeding during the first

month of life is associated with decreased TLR mediated cytokine production. Decreased TLR

mediated cytokine production helps reduce the level of cytokines in the blood, promoting

glucose homeostasis and normal insulin secretion by the pancreas. Immune modulation by breast

milk is TLR-specific. This is because gut microbiota, influenced by breast milk, suppress

cytokine production regulating TLRs. Promotion of early life innate immune maturation might

be one of the mechanisms by which breastfeeding protects against subsequent disease. More

insight into the effects of breast milk on the developing immune system is needed for the

generation of immune modulatory strategies that use breastfeeding to prevent subsequent

disease.

A better understanding of the key physiologic differences between breastmilk and infant

formula obtained from studies may provide useful insights into the development of new infant

formulas to better emulate the effects of HM on the development of infant intestinal microbiota.

Further research is needed to study the highly selective Bifidobacterium infantis strain. Also,

further research is needed when it comes to infant formula development. Because human milk

varies among individuals and as the infant ages, it is difficult to mimic its beneficial components

in infant formula such as live cells, bioactive compounds, and its exact nutritional composition.

47
When adding ingredients found in human milk, infant formula manufacturers need to consider

the form of the molecule of each nutrient thats added, bioavailability, other compounds that the

nutrient depends on for absorption and proper utilization, and the amount that is added in order

to avoid toxicity.

This paper suggests the potential for breastfeeding to protect against the onset of T2DM

with the gut microbiome functioning as a mediator. Sufficient evidence supports the link

between breastfeeding and the microbiome, the microbiome and T2DM, and breastfeeding and

T2DM; there are no studies that prove the link between all three factors. This paper provides

enough evidence to support the need for a prospective cohort study that would potentially follow

a group of individuals from infancy to adulthood and assess the microbial development along

with the onset of T2DM later in life. In general, further research on the gut microbiome in

individuals with T2DM can provide evidence for the potential benefits that treating the gut

microbiome can have on preventing or reversing the disease.

48
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