ABSTRACT

The present study is carried out to evaluate the interaction of cortisol secretion in type 2
diabetic patients. This case control study is carried out on a total of 70 subjects out of which
35 were diabetic male patients and 35 were non-diabetic individual males who were carefully
selected. All subjects aged between25-50. Physical parameters of the study include age and
body mass index (BMI) whereas biochemical parameters to be assessed include HbA1c and
secretion of cortisol in control and diabetic patients. It was analyzed that individuals aged
above 30 have more chances of occurrence of disease. BMI has no effect on prevalence of
diabetes. As far as, biochemical analysis is concerned it is found that in normal individual’s
cortisol secretion is not much high however it is seen to be vary in diabetic patients. The
present study depicts that cortisol secretion does not have any effect on diabetic patient.
Secondly, HbA1c is glycated. Hemoglobin that act as projection marker in diabetic patients.
Its level tends to be increased in the following research study in diabetic patients but in
control subjects its value (<40(mmol/mol)) remained normal. Therefore, it is concluded that
cortisol secretion has no effect on incidence of diabetes however it is age dependent disease,
prevalence is more in old age individuals.

Key words: Diabetes, Cortisol, HbA1c

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INTRODUCTION

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1.0 INTRODUCTION

Extensive and in-depth studies have shown that increased cortisol level present in blood are marked as
the significant projection factor for cardiovascular disease, such as diabetes mellitus. Glucose is one
of the prime energy sources for human body. It act as a fuel for cells and circulates throughout the
blood (Piero, Njagi et al. 2012).The regulation of blood glucose concentrations is owned to a
pancreatic hormone insulin. The hormone gets bind to peripheral portion of cell membranes to its
specific receptor sites. The main function of this hormone is to permit the entrance of glucose into
respiratory cells and tissues by mean of entailed channels (Kibiti 2006). Through the process of
glycolysis insulin triggers the conversion of one molecule of glucose into two molecules of
pyruvate /pyruvic acid. Moreover, it is also involve in glycogenesis and lipogenesis (Njagi 2006). 
Glucagon acts antagonistically to insulin, therefore above mentioned metabolic events are opposed by
this hormone. Glucose remains in the blood instead of entering the cells, when the levels of glucose
are below the threshold degree. This would lead to hyperglycemia by withdrawing water from the
cells. The excess amount of sugar present in urine is excreted out, this condition is called glycosuria.
That’s why patients of diabetes complain constant thirst, drinking large amounts of water help to get
rid of this condition. Due to deficiency of insulin, hyperglycemia may prolong as the result body cells
get deplete of glucose. This enforces the cells to find some alternative resources of energy. For this
purpose, the cell utilizes fatty acids stored in adipose tissues. The fats are not sources of fuel for red
blood cells, kidney cortex, and brain. There is no β- oxidation in the mitochondria pathways of red
blood cells. To use energy for such cells and tissues, the acetyl-coA resulting from catabolism of fatty
acids is diverted to ketogenesis to generate ketone bodies which can serve as alternative sources of
fuel for such cells and tissues. These ketone bodies are also passed to the urine, leading to ketonuria
which characterizes diabetes mellitus. The generation of ketone bodies in blood results in ketosis.
ketone bodies lower blood pH as they are acidic in nature therefore, leading to acidosis. This
combination of ketosis and acidosis would lead to a condition called ketoacidosis. It left untreated,
ketoacidosis will lead to coma and eventually death (Belinda 2004).
Diabetes mellitus(DM) is the most common endocrine disorder affecting more than 100 million
people worldwide (6% of papulation). The prime cause of DM is deficiency or ineffective pancreatic
insulin secretion resulting in elevated or decreased blood glucose concentration. It also affects many
other body systems, particularly blood vessels, eyes, kidneys, heart and nerves (Ismail and Yaheya
2009). Chronic hyperglycemia diabetes is related to long-term organ damage, dysfunction and failure,
more specifically the eyes, kidney, nerves, heart and vessels of the blood. Various pathogenic
mechanisms are involved in development of diabetes. They may range from autoimmune distortion of
pancreatic β-cells with consequents insulin deficiency to abnormalities that would result in insulin
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resistance. The deficiency of insulin in target tissues provides basis for carbohydrate, fat and protein
metabolism abnormalities in diabetes. poor insulin action results inadequate secretion or decrease
tissue response to insulin at one or more junction in the complex pathway. The prime cause of
hyperglycemia is impairment of insulin secretion and insulin action defects often coexists in the same
patient (care 1997). Age is an important factor, the frequency of diabetes mellitus increases with age.
Impaired renal function and increased sympathetic nervous system activity and post receptor
impairment of fat tissues occur due to glucose mediated insulin secretion decrease and glucose
tolerance impairment. However, in different studies, it has been observed that cortisol secretion does
not change by age rather a decrease has been observed in its catabolic reactions. Diabetes mellitus has
been classified into insulin-dependent diabetes mellitus non-insulin dependent diabetes mellitus (5-
10) percent of total diabetes results from autoimmune destruction of pancreatic β cells causing
absolute loss of insulin secretion. It is autoimmune disorder which is characterized by inflammatory
reactions in islets of Langerhans, followed by selective destruction of insulin secretion cells. 90-95
percent of people with diabetes may suffer from type 2 diabetes. This type of diabetes is caused by
relative insulin deficiency and insulin remissness to perform its task. Individuals suffering from type 2
diabetes patients are mostly overweight because in response to increase fat content in the body,
insulin resistance develops (Arora, Ojha et al. 2009). A number of genetic and environmental factors
interact to generate a heterogeneous and progressive disorders with different levels and degrees of
insulin resistance and pancreatic β cell dysfunction. When β-cells are cells not able to secrete higher
insulin to overcome insulin resistance, impaired glucose tolerance progresses to type 2 diabetes.
Insulin resistance and impaired glucose tolerance more repeatedly results due to overweight and
obesity (Jothivel, Ponnusamy et al. 2007).

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Figure 1.1 differences between diabetes pancreas and type 2 diabetes pancreas their insulin
production, glucose level in blood and their movements into cells. There are many other factors
such as peptide 1 ( GLP1) hyper glucagon anemia, and increased levels of other counter regulatory
hormones also contribute or involve in insulin resistance and decrease insulin secretion and type 2
diabetes hyperglycemia (Drucker and Nauck 2006). C Cortisol high secretion is related to increase
blood pressure and several metabolic disturbance resulting in abnormal obesity, insulin resistance and
reduce secretion of HDL cholesterol that can lead to diabetes (Connell, Whitworth et al. 1987).
Approximately 50% of all patients have raised cortisol levels. This probably involves the increase
secretion of hypothalamic CRH leading to increase of ACTH and cortisol secretion (Holly, Amiel et
al. 1988). Intense phobic anxiety in scale of the SCL-90 may result increase cortisol levels in
controlled diabetic patients. Cortisol is a glucocorticoid hormone which is produced by zone
fasciculate of adrenal cortex and same to other body hormones of the adrenal cortex cortisol
is a derivative of cholesterol. Stress, either psychological or due to some other reasons
(trauma, surgery, injection, anesthesia, and hypoglycemia) and depression trigger the
secretion of hormone of the adrenal cortex of hypothalamic CRH in the brain that eventually
leads in production of ACTH by pituitary glands, which act on the adrenal glands to stimulate
the

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production of many hormones, including cortisol (Rossi, Tauchmanova et al.
2000). Hormones which is involve in glucose high production is insulin, amylin, glucagon, incretins,
epinephrine, cortisol, growth hormone, polypeptide, leptin, ghrelin, and
adiponectin. .Insulin (retards the production of glucose from glycogenin the,liver).Amylin
leading to delayed stomach emptying and lowers glucagon levels).Glucagon (It converts stored glyco
gen into glucose in muscles and liver and also decreases the assimilation of glucose by the liver),
GLP-1 (glucagon like peptide) and GIP (glucose dependent insulin tropic polypeptide) that
are produced by intestine in response to food absorption and helps in producing and
enhancing the release of insulin by increasing the production of Integrin β7 cells and delays
the release of glucose. They inhibit the release of glucagon but in case of diabetic patients
low GIP levels are observed and their β-cells do not respond to GLP. Cortisol increase blood
sugar concentrations by increasing the insulin resistance in muscles and conversion of
glycogen and protein into glucose inside liver. By increasing muscle insulin resistance and
converting glycogen and protein to liver glucose, cortisol elevates blood sugar levels.
Epinephrine is secreted from the ends of the nerves and produced in adrenal gland. It results
in glucose production by acting on liver. It also inhibits insulin release, which causes
increased blood sugar levels. Cortisol promotes the production of glucose from stored
glycogen and protein in liver. It also makes the muscle cell insulin resistant, which leads to
reduce glucose entry into cells and leads to increased blood sugar levels. Cortisol, which
causes gluconeogenesis, breakdown of fat and protein, and mobilization of extrahepatic
amino acids and ketone bodies, suppresses insulin secretion from beta pancreatic cells (Ziaja,
Cholewa et al. 2008).One of the most dominant hormone in the human body is glucocorticoid cortisol
(hydrocortisone). Through multidirectional action, it prepares the body how to handle body with
physical and mental stress. Cortisol also affects the metabolism of proteins, carbohydrates and fats
and contributes to the upregulation of water and electrolyte balance, blood pressure, body
temperature, bone mineralization and immune response (Vaseghi, Hajhashemi et al. 2014).cortisol
contributes in the perception of mood and behavior, appetite and pain. It triggers fight and flight
mode in organisms by affecting the functioning of many organs and systems. Also regulates the use of
dietary nutrients by increasing gluconeogenesis and increasing lipolysis in adipose tissue.
Furthermore, it increases protein synthesis in the liver and restricts their formation in muscles and
epithelial cells.

It also balances water and sodium retention, controls adrenaline production in the adrenal medulla,
inhibits inflammatory processes and also affects the psyche, often leading to euphoria or depression.
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Figure 1.2 pathways how stress and other factors effects cortisol production their effect on
human body and conversion into type 2 diabetes.
Glucocorticoids retards inflammatory response specifically cortisol suppresses arachnidonic acid
production and secretion a projection marker for a number of inflammation mediators. Due to
stabilization of lysosomes, decrease in the number of circulating T4 lymphocytes and decrease in the
production of key immune response mediators increased levels of glucocorticoids hormone can lead
to the suppression of the body’s immune response eventually, reducing the ability of the body to
recognize and defend itself from foreign entities such as bacteria and viruses. Insomnia as well as
several mood swings also participate in increasing cortisol concentration (Ziaja, Cholewa et
al.2008).Properly composed diet provides all nutrients derived from plants and animal
production. Cortisol increased the metabolism endogenous cholesterol production beings with
the condensation of three acetyl Co A molecules. Furthermore, the metabolic reactions depend
on the presence of the number of enzymes and the inherence of vitamin B3.

As the result of a triple enzymatic hydroxylation of cholesterol, which is released into the blood
by passive diffusion process level of cortisol rises (Sapolsky, Romero et al. 2000). The synthesis
and metabolism of cortisol including effects of B vitamins (B3 and B5). The metabolism of
cortisol such as tetra hydro cortisol is biologically inactive and occurs in its conjugated form as
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glucuronic acid. It is inactivated in the liver after conjugation with glucuronic acid that has been
excreted in the urine. Occurs mainly in the liver, where it is converted to tetra-hydro-metabolites
(THMs) via reductases, which are then excreted through urine. Before it reaches the target cell
receptors under microsomal 11β-hydroxysteroid dehydrogenase, this hormone may undergo
alterations in the organism which reversibly deactivates it to cortisone (Fink 2010).Cortisol circulates
in three forms in the blood such as free cortisol (5 percent), protein-bound cortisol, and metabolites of
cortisol. The concentration of plasma cortisol in the blood is 10 to 15 ug/dl percent unbound (free)
cortisol present only which is the physiologically active hormone. This amount of cortisol excreted in
the urine. Approximately 90 percent of cortisol is bound to cortisol binding globulin (CBG), also
known as transcortin, and albumin. CBG has a high affinity for cortisol but is present in small
amounts. Various, dietary and endogenous factors affect the maintenance of threshold concentration
of glucose in human blood. Cortisol acts on the amount of glucose by activating hepatic glycogen
stores, reducing glucose oxidation, stimulating lipolysis, and intensifying gluconeogenesis from
amino acid severity. Cortisol affects collagen synthesis, however, increasing its amount in the blood
stream, caused by mental stress or excessive physical effort, leads to a number of modifications within
the structure of osteoblast and concentrations of chemical substances involved in the bone remodeling
process. These alterations brought about disturbances in bone mineralization and inhibition of
collagen synthesis (Reynolds, Labad et al. 2010). Cortisol enhance blood vessel and heart tissue
sensitivity to noradrenaline, vasopressin and angiotensin II, eventually resulting in increased blood
pressure.it also effects water and electrolyte balance, increasing water and sodium retention. Cortisol
prevent overheating of the organisms by acting on body thermal regulation mechanism (Reynolds,
Labad et al. 2010). Hydrocortisone affects the immune system showing anti-inflammatory and
immune suppressive effects by reducing the secretion of pro inflammatory cytokines, decreasing the
migration of white blood cells to inflammation sites and inducing cell apoptosis. This stimulates the
body’s defensive response to injuries, including those that occur during training and starting sport.

Cortisol influences neuronal plasticity and neuro degenerative processes by acting on neurons and
glial cells within the central nervous system. It also modifies the mood and behavior as well as the
perception of pain, which is particularly important in mobilizing players at the start time (Elenkov and
Chrousos 2002). Cortisol known as stress, fighting hormone, prepares athletes to increase their effort.
However, its excess leads to catabolic reactions that have a negative effect on the organism. The
levels of cortisol as a catabolic hormone remains in dynamic equilibrium with anabolic hormones. It
has been shown that high concentration of cortisol (C) inhibit testosterone (T) synthesis in the body,
contributing to disorders in the quantity ratio of these hormones. Estimated C / T is an indicator that
reveals athletes exhaustion and over training (Obmiński and Tourism 2009). In adult’s man’s studies,
higher morning fasting plasma cortisol levels, acute imeasurement of cortisol secretion is linked with
higher blood pressure, plasma glucose and triglyceride concentration and lower birth weight. Children
and adolescents with lower birth weight were also reported to excrete more cortisol or its urine
metabolites (Harland, Watson et al. 1997). These studies have led to the hypothesis that events in
daily life permanently changed or program cortisol secretion, and that this along with increased
obesity leads to a high prevalence of metabolic syndrome and cardiovascular disease in adult life. We
investigated a group of men of known birth, weight to test this hypothesis by characterizing
abnormalities of cortisol secretion in relation to metabolic syndrome, obesity, and birth weight
characteristics. Glucocorticoid secretion has been suggested as a possible connection between insulin
resistance and metabolic syndrome in patients with type 2 diabetes, associated abnormal
characteristics includes (hypertension, obesity, coronary heart disease, hyperlipidemia and type 2
diabetes). In patients with type 2 diabetes the hypothalamic pituitary adrenal (HPA) axis secretion has
been extensively investigated in recent years (Cameron, Kronfol et al. 1984).The presence of chronic
abnormalities of type 2 diabetes (i.e., macro angiopathy,retinopathy, and neuropathy) has been
associated with the activity of the HPA axis and has recently been reported to be associated with the
severity of several clinical parameters of diabetes and cortisol secretion in type 2 diabetic subjects
with normal HPA activity Hypothalamic Pituitary Adrenal (HPA) axis, an axis that secretes cortisol
along our brain. This would result in a series of events down the HPA axis that ultimately lead to the
secretion of cortisol from the adrenal gland hormones produced by the hypothalamus(Roy, Roy et al.
1998).

Endogenous hyper-cortisolism (EH) Cushing's syndrome / illness. Cushing's syndrome (CS) Diabetes
mellitus is common in patients with Cushing's syndrome. This syndrome affects glucose tolerance
range and may lead to insulin resistance. However elevated cortisol levels may be associated with
type 1 diabetes. (Arnaldi, Angeli et al. 2003).The action of cortisol in patients with type 2 diabetes in
associated with hippocampus atrophy. It was observed that patients with type 2 diabetes showed
reduced cortisol activity, in combination with reduced hippocampal volume (Etxabe and Vazquez
1994). The occurrence of chronic metabolic complications such as insulin resistance, diabetes
mellitus, dyslipidemia, cardiovascular disease and bone fragility leads to Hyper-cortisolism (Mancini,
Kola et al. 2004). Diabetes is considered a common complication of chronic exposure to excess
glucocorticoid and is an important contributing factor to morbidity and mortality in patients with EH.
Overall EH mortality enfolds two times the general population, it has been observed in various
clinical setting that diabetes, hypertension and uncontrolled hypercortisolism are significant prediction
factors of overall mortality. The patients, who achieve disease remission after many years of
hypercortisolism, have an increased risk of death. Most deaths occur in patients with persistent
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hypercortisolism. (Pagano, Cavallo-Perin et al. 1983).While a Spanish research revealed an incidence
of 2.5 cases per one million persons per year. A recent large scale retrospective survey conducted by
Bolland in New Zealand showed that the prevalence of all forms accounts for 79 cases per million
and the incidence was 1.8 per million per year (Abdelmannan, Tahboub et al. 2010).Glucocorticoid
induced diabetes is just like type 2 diabetes because glucocorticoids impair the glucose metabolism
mainly by increasing insulin resistance in the liver with increased production of basal glucose and in
adipose and skeletal tissues with impaired glucose (Nicod, Giusti et al. 2003).T2D is multifactorial
and polygenic diseases including several environmental factors (i.e., exercise and diet) and an
underlying genetic pre-disposition influence the phenotype. The phenotypic depression has
physiologically a centrally-hyper-activated HPA axis and thus high cortisol levels. Hypercortisolism
is the most frequently studied finding , and is generally accepted that CRH also increases in
depression(Gold, Chrousos et al. 1984). Insulin resistances, the common thread between obesity, the
metabolic syndrome, and type 2 diabetes mellitus, is defined as the impaired ability of insulin to
control nutrient partitioning in target organs. In adipose tissue, insulin fails to restrain lipolysis and
increase glucose uptake; in liver, to inhibit hepatic gluconeogenesis and glycogenosis, and in muscle,
to induce glucose uptake.

A critical function of GCs is to liberate energy substrates (i.e, glucose, amino acids, and fatty acids
[FA]), and thus ensure their availability for mitochondrial oxidation in fight-or-flight responses. Thus,
GCs elevates muscle protein breakdown, adipose tissue lipolysis, and hepatic gluconeogenesis.
Moreover, it reduces glucose utilization. The overexposure of Chronic GC changes composition of
body, which includes more deposition of fat in adipose tissue, and impairs and insulin action
metabolism resulting in hyperglycemia and dyslipidemia (Rosmond 2003).

REVIEW OFLITERATURE
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2.0 REVIEW OFLITERATURE

The concentration of IGF-1 (insulin like growth factor 1) and DHEA-S (Dehydro-epiandro-sterone-
sulfate) expressively decrease while on the other hand serum cortisol and cortisol DHEA-S have
higher concentration in both male patients of diabetes and non-diabetes. These hormones also vary in
diabetic when non diabetic male patient have MS (multiple sclerosis) IGF-1 individually connected
with waist margin blood pressure and fasting blood glucose. The levels of IGF-1 is reduced and
cortisol DHEA-S levels increased along with insulin resistance in adult male with diabetic MS (El-
Eshmawy, Hegazy et al. 2011).

In several clinical measures the degree of severity is associated with cortisol concentration in
type 2 diabetes. there is a positive connection between metabolic disturbance and cortisol
levels that are within the accepted normal range. Increase in the levels of salivary cortisol
above the set spectrum affects the patients by various ways. Cortisol hormone is related to
post prandial urinary acid, glycosylated hemoglobin, systolic and diastolic blood pressure in
patient with type 2 diabetes. cortisol levels increased above to normal range is based on
cortisol sampling time just prior to a stand lunch, the cohort was divided into tertiles. Cortisol
level also related with the relative abdominal mass when patients with marked glycosuria
were excluded (Oltmanns, Dodt et al. 2006).
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Higher cortisol concentration is associated with patients of type to diabetes. They support role of
morning serum cortisol in glucose metabolism along with individuals of type 2 diabetes. morning
serum cortisol is also linked with increase FPG (fast plasma glucose) and lower β-cell function among
individuals without type 2 diabetes and increase FPG and HbA1c (hemoglobin A1c levels) in
participants with diabetes (Ortiz, Kluwe et al. 2019). High cortisol infusions of glucose synthesis is
find by the result of gluconeogenesis (break down of glucose) and other hormone is locked. Diabetes
pathophysiology is associated with the increase cortisol. Especially it is a dependent factor of
hyperglycemia. The impact of cortisol on these factor is a result of the immediate intervention of
cortisol on hepatic gluconeogenesis (Khani and TAYEK 2001).
High secretion of cortisol is associated with obesity. Effect of cortisol on gluconeogenesis
and other insulin parameters resistance are determined in normal subjects. The pituitary
pancreatic infusion balance insulin, GH (growth hormone) and glucagon concentration within
the diet range by the increase of the level of cortisol.

The regulation of cortisol hormone in human body increased with the increase of GP by
stimulating gluconeogenesis. A miner increase in serum cortisol may disturbed the normal
glucose metabolism that cause the metabolic syndrome (Khani and TAYEK 2001). Higher
fasting cortisol concentration are not associated with current growth performance in individual
cognitive domains according to age-adjusted analyses. On the other side increased fasting cortisol
concentration are associated with greater estimated cognitive decreasing and the working and
processing speed of memory mood independence education, metabolic variables and heart disease. In
old age peoples high morning cortisol concentration with type 2 diabetes are related with age-related
cognitive change. Strategies targeted at lowering cortisol action may be useful in improving cognitive
reduction in individuals with T2D (Reynolds, Labad et al. 2010).
Homocysteine is positively associated with psychoticism (refers to a personality pattern by
aggressiveness and interpersonal hostility) and negatively to the act out hostility and
somatization (form of mantle illness) in controlled diabetic patients. In controlled diabetic
patient’s cortisol is negatively associated with extraversion and positively with phobic
anxiety. Homocysteine and cortisol are also associated to individuals and state psychological
factors in patients with T2D mellitus (Kontoangelos, Papageorgiou et al. 2015).
lower aged patients sustain higher prevalence of type 2 diabetes mellitus CHD (coronary
heart disease) osteoporosis and ruptures with respect to NSA (national security agency).
Intermediate phenotypes also showed higher prevalence with heart diseases and T2D with
respect to NSA. Increasing subclinical hypercortisolism in adrenal adenomas is associated
with high prevalence of adverse metabolic and
i cardiovascular outcomes, freely of other
possible risk factor (Coiro V, Volpi R,1995).
Increase cortisol concentration is associated with important metabolic variations of intermediate.
Metabolism involved in abdominal obesity, insulin resistance and lower concentration of HDL-
cholesterol that involve in diabetes. the function of glucocorticoids in glucose homeostasis is linked
between hypercortisolism and type 2 diabetes. effect of cortisol secretion and action yet unknown
contacts between insulin resistance and other metabolic syndrome features (Di Dalmazi, Pagotto et al.
2012).

preeminent plasms cortisol concentration is linked with high fasting glucose and total
cholesterol levels. The association between HPA axis activation and the metabolic syndrome
are present between peoples with type 2 diabetes. The connection of elevated plasma cortisol
with high prevalence of cardiovascular disorder is independent conventional risk factor. The
role of cortisol in the pathogenesis of ischemic heart disease merits is further exploration
(Reynolds, Labad et al. 2010).
Hypothalamic pituitary-adrenal (HPA) axis emission in patients with type 2 diabetes is determined by
serum cortisol and late-night salivary levels of cortisol. The existence of chronic abnormalities of type
2 diabetes such as micro-angiopathy, retinopathy and neuropathy also associate with the activity of
the HPA axis and the assemble between the degree of severity of several clinical interferences and
cortisol secretion in type 2 diabetic individuals with normal HPA exercise has lately be noted
(Chiodini, Adda et al. 2007).
In type 2 diabetic individuals hypothalamic-pituitary-adrenal activity is well developed in diabetic
patients. complications and the concentration of cortisol secretion is assosiate with the existence the
number of diabetes complications. Cortisol secretion also linked with difficulties and metabolic
control of diabetes in type 2 diabetic individuals (Castillo-Quan, Herrera-González et al. 2007)
Depression shows the increase risk of progressive insulin resistance and incident type 2 diabetes
mellitus. And the linked between stress and diabetes is not too much clear due to differences in study
parameters and in forms and ascertainment of stress. Different biological parameters involved in find
out the connection between stress and physiological functions (hypothalamic–pituitary–adrenal (HPA)
axis, immune systems and autonomic nervous. The HPA axis is a strongly regulate the system that
represent the body different mechanisms and responsible for acute and chronic stress. Association of
depression with cross-sectional and longitudinal alterations in the daytime cortisol curve. Diurnal
cortisol curve Flattening is also associate with insulin resistance and type 2 diabetes mellitus (Joseph
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and Golden 2017).
Underweight patients with type 2 diabetes have less homocysteine (non proteinogenic α
amino acid) concentrations with associated to healthy controls. [G.Marchesini,2002]. Obese
patients with type 2 diabetes presented greater homocysteine standards. when compared to
healthy controls. More over homocysteine and cortisol are associated with characteristic and
state psychological factors in patients of T2D mellitus (Chiodini, Adda et al. 2007).

Outputs of urinary-free cortisol (UFC) in diabetic patients may or not associated

with complications. Diabetic patient’s presents the imbalance of the hypothalamic-pituitary-
adrenal (HPA) axis and also have slight imbalance of the HPA axis. This imbalance is
associate to extensive period of diabetes (Roy, Roy et al. 1998).
Patients with bipolar disorder (depressive illness) are associated with type 2 diabetes mellitus
and the association appears not too much related with co-morbidity. These two disorders have
same pathophysiologic origins and share common genetic associations with epigenetic
procedures. Bipolar patients with co-morbid type 2 diabetes mellitus have a simpler passage
of bipolar illness and are more refractory to treat the bipolar disorder. The type 2 diabetes
mellitus often remain under-treated. Control of diabetes not as good as the rate of diabetes
complications is larger associated with diabetic patients and without bipolar disorder. So
bipolar patients with type 2 diabetes mellitus have significantly greater CV morbidity and
mortality (Calkin, Gardner et al. 2013).
Hormone (testosterone, cortisol) association with other hormones and among masculine competitors
and far less so between woman competitors. The concentration of testosterone and cortisol increase
during the anticipation of games. Cortisol shows closely association with player evaluations of
whether the adversary was more of a challenge than anticipated and was negatively associate with
loss (Bateup, Booth et al. 2002).
Cortisol patterning differs between healthy individuals and with diabetes individuals
according to cross-sectional evidence. For the first time the imbalance of cortisol in regular
life can predispose people to new-onset diabetes advanced in life independently of traditional
risk issues(Hackett, Kivimäki et al. 2016).
People with type 2 diabetes there is an association among HPA axis activation and features of
the metabolic syndromes. Hair cortisol also linked with raised glycated hemoglobin (HbA1c),
chronic contact and higher cortisol is linked iwith dysglycemia. High plasma cortisol is
associated with higher occurrence of ischemic heart disorders independent of conventional
risk factors. Higher plasma cortisol concentrations also linked with increasing fasting
glucose and total cholesterol concentrations. (Ortiz, Kluwe et al. 2019).
males have larger breast circumference and a less BMI (Biomass index) as compare to female. Both
male and female have similar glycemic control, but in males there are comparatively greater diastolic
blood pressure and less HDL-cholesterol concentration. Male also have greater heart issues.

Occurrence of Cortisol concentration enhanced with age but different depending on the sex, liquor
consumption, tobacco marriage status or the lowest instructional standard attained. In male’s cortisol
levels were significantly same as compare to higher waist and higher BMI but not different
significantly depends on the body fat concentrations. Cortisol not associated with obesity in female fat
body. Variables of cortisol and metabolic syndrome high plasma cortisol were significantly matched
with high fasting glucose but not with diabetes or HbA1c. A significant positive relationship among
cortisol and total cholesterol was detected, but not significant association was observed with HDL-
cholesterol (Rosmond, Dallman et al. 1998).The fasting cortisol concentration in individuals was
high and was much higher than the concentration that observed in without diabetic patients.
People with type 2 diabetes attained more stress on plasma cortisol(Harden, Bruehl et al.
2007).
Type 2 diabetes mellitus is the result of interaction among genetic and environmental factor correlated
to heterogeneous and permanent β-cell pancreatic dysfunction. Overweight and obesity is an
essential growth factor of insulin resistance and decrease glucose sensitivity. Nonfunctioning
of β neurons to release enough glucose which turns in type 2 disease. Difficulties in other
hormones for example less secretion of in cretin glucagon-like peptide 1 (GLP-1)
hyperglucagonemia, and increased concentrations of other regulatory hormones also
participate in insulin resistance, less insulin excretion, and hyperglycemia in type 2 diabetes.
Higher cortisol is also linked with metabolic variations of intermediate metabolism causing
abdominal obesity, low HDL cholesterol and insulin resistance levels that change into
diabetes (Di Dalmazi, Vicennati et al. 2012).
Rats exposed to glucocorticoids in utero have higher plasma glucocorticoid concentration ,
which are linked with lower concentration of glucocorticoid receptors in pituitary gland and
brain which may damage negative feedback control of CRH and ACTH excretion
glucocorticoids purposes in glucose homeostasis and explain the co relation among
hypercortisolism and type 2 cancer (Levitt, Lindsay et al. 1996).
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Glucocorticoid receptor expression in male then dexamethasone suppression of plasma
cortisol is expected to be decrease in males with decrease birth weight, but it was maintained.
fasting plasma cortisol increase in low birth weight male without dexamethasone but was not
vary after dexamet has one of administration ,the incremental outcome of dexamethasone
may be higher As an alternative of dexamet may not cross the blood-brain barrier properly at
minor doses in person (Meijer, De Lange et al. 1998).

Elevated plasma cortisol will lead from higher drive to CRH, ACTH and cortisol excretion
from higher centers as an increase in plasma cortisol when stressed on first testing. Cortisol
secretion increase from adrenocortical sensitivity to ACTH. Male with the cluster of
cardiovascular risk factors contains low birth weight and the adult metabolic syndrome and
activation of the HPAA. That relationship among low birth weight and subsequent
cardiovascular disease and may suggested novel therapeutic strategies to decrease
cardiovascular effects. Males with cluster of cardiac threat include low birth weight and
adult metabolic syndrome has HPAA activity (Reynolds, Walker et al. 2001). Cortisol is a
stress hormone and also linked with learning of individual’s. Its effects on memory, learning
and behavioral level in males and females. In this check the effect of a single cortisol dose
(30 mg) on the hemodynamic association of fear conditioning. Cortisol impaired electro-
dermal signs of learning in males, while no conditioned SCRs emerged for the females
independent of treatment. According to fMRI cortisol decrease activity for the CS+ > CS−
comparison in the anterior cingulate, the lateral orbitofrontal cortex and the medial prefrontal
cortex in males. On the other hand increase in these regions under cortisol checked out in
females. In addition, decrease the concentration of cortisol the habituation in the CS+ > CS−
contrast in the dorsolateral prefrontal cortex independent of sex. So cortisol also response to
the electric shock by increasing the activity of anterior and posterior cingulate. These shows
that humans cortisol generally influences prefrontal brain activation throughout fear
conditioning and that these effects appear to be modulated by sex(Stark, Wolf et al. 2006).
The action of insulin on glucose manufacturing and utilization per number of monocyte and
erythrocyte insulin receptors was reduced. These indicate that the cortisol-induced insulin
resistance in male is due to the reduce in both hepatic and extrahepatic sensitivity to insulin.
Insulin bind with monocytes and erythrocytes reflects insulin binding in insulin-sensitive
tissues, that decrease in insulin action explained on the basis of a post receptor defect(Rizza,
Mandarino et al. 1982). Diabetic individuals with chronic complications were found to be
more frequent in male and present high A1C concentration and long interval of disease with
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respect to diabetic patients and without chronic complications. Age, A1C, and duration of
diabetes directly linked with cortisol secretion in type 2 diabetes individuals with normal
HPA activity. Hypothesize that these variables might be responsible for increase cortisol
secretion in diabetic individuals with chronic complications.

Increased cortisol secretion may participate in metabolic control of diabetic and insulin
sensitivity thus inducing a high occurrence of chronic diabetic complications. 80% patients of
Cushing’s syndrome increased albumin excretion. In type 2 diabetes individuals HPA activity
is increase in patients with chronic complications and the degree of cortisol excretion is
directly linked with the occurrence and the number of diabetes complications (Chiodini,
Adda et al. 2007). Glucocorticoid Concentration change throughout the day. The reduced
rates of glucose intake resist insulin secretion. Cortisol infusion also higher postprandial
palmitate appearance as well as palmitate, alanine levels suggesting stimulation of lipolysis
beta-hydroxyl butyrate, ketogenesis, and proteolysis. The circadian changes in cortisol levels
on physiologic importance in normal humans (Dinneen, Alzaid et al. 1993).Stress in teleost’s
(Fish form) is well studied because of its interaction with growth, reproduction, immune
system and ultimately fitness of the animal. Cortisol is reliable indicator of stress and is
considering the main stress hormone. At the start cortisol is measured in blood, but now
evolving towards lower invasiveness (disease or condition that has ability to spread) and
allow repeating measurement of cortisol after some time interval. In past cortisol level
determination in fishes, present new changes in blood, whole body and eggs as matrices for
cortisol measurement, notably mucus, faeces, water, scales and fins. On the other side new
analytical tools are being established to increase specificity, sensitivity and automation of the
measure. The founding principles of these techniques and also there introduce potential as
continuous monitoring tools (Sadoul and Geffroy 2019). Hypothalamus–pituitary–adrenal
axis in response of stress is activated and glucocorticoids excreted. These hormones exert
multiple effects in the periphery nervous system and on brain. As such they improve memory
consolidation, but at the same time the ability to retrieve earlier learned information lower.
Glucocorticoids appear to interact with memory(short-term) working. High glucocorticoid
concentration as a result of endocrine or psychiatric disorders or as part of age-linked
variations in the hypothalamus–pituitary–adrenal system, typically they have a negative
effect on memory. In parallel, structural changes are detected in the hippocampus (is a small,
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curved formation in the brain that plays an important role in the limbic system also play role
in memory formation) and the prefrontal cortex. The effects of glucocorticoids on CNS will
ultimately lead to development in the treatment of psychiatric and systemic diseases which is
cause by hypothalamus–pituitary–adrenal hyper- or hypo-activity(Wolf and Metabolism
2006).

Mental stresses as compare to physical stress participate in the formation of adverse

alternation in hormonal balance. Higher concentration of cortisol (which is a stress hormone)
leads to negative changes in body functioning. Endocrine difference decrease using a suitable
way and quality of nutrition. Various nutrients is dependent on gender, age, kind of work and
lifestyle. Variety of manufacturing food permits the use of a varied diet, taking into account
individual needs and preferences of the individuals. It is very essential to preserve acceptable
nourishment (Stachowicz, Lebiedzińska et al. 2016). Stress has both beneficial and harmful
effects. The beneficial effects of stress include preserving homeostasis of cells and species,
which leads to sustained existence. The harmful effects of stress may obtain more attention or
recognition by an individual due to their role in different pathological conditions and
disorder. Different factors such as neuroendocrine mediator’s hormones, neurotransmitter
sand peptides are involved in the body's response to stress. Number of diseases originates
from stress, especially if the stress is prolonged. Stress cause number of disorders and treat
the patient therefore using both pharmacological (medications and/or nutraceuticals) and non-
pharmacological (change in lifestyle, daily exercise, healthy nutrition, and stress reduction
programs) therapeutic introduced (Yaribeygi, Panahi et al. 2017).

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MATERIALS AND METHODS

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3.0 MATERIALS AND METHODS

3.1 SOURCE OF DATA

In order to conduct the research, the subjects were recruited from Jinnah hospital Lahore and
Sheikh zayed hospital Lahore. This case control study carried out on a total 70 subjects.35
with diabetes and 35 without diabetes were selected. The purpose of study was to identify
the interaction of cortisol secretion in type 2 diabetes patients of age 25-50. For this
approximately 0.1 ml of serum is required per duplicate determination. Collected 4-5 ml of
blood into an appropriately labeled tube and allowed it to clot centrifuge and carefully
remove the serum layer. Store at 4oc for up to 24 hours or at -10oc or lower if the analyses
are to be done at a later date.

3.2 ETHICAL CONSIDRATIONS AND CONSENT PROCESS

The study was approved by the Ethical committee of hospitals and detailed histories of male
were obtained from both hospitals. From each participant male including normal and diabetic,
informed consent was taken. Moreover, the blood samples were also collected from all
involved participants.

3.3 INCLUSION CRITERIA

The case participants involved in the research were male aged between 25 to 50 years with
the diabetes. The control participants includes normal healthy male. Both cases and controls
confirmed by HbA1c (Glycosylated Hemoglobin) Test.

3.4 EXCLUSION CRITERIA

The entire male with age less than 25 years, known history of metabolic disorders, diabetes
mellitus, Cushing syndrome and drug abuse was completely excluded from the study. Each of
the women from the selected samples was assessed for only one time.
3.5 STUDY DESIGN AND PROTOCOL i
The study design used in this study is the case control study and once the synopsis was
approved, the data and samples were collected during the time of 6 months. The data utilized
for the study is collected through the primary data resources; therefore, the research approach
used in this study is quantitative in nature. The primary data was particularly gathered
through the questionnaire, experimental techniques and observation of the information
provided by the participants. It was insured that all the participants of the study were
provided with the detailed information related to the research and informed consent was
taken for the study. The information gathered from the patients including name, bio data,
address, age, history of diabetes mellitus. The physical examination information such as
weight, height, BMI was also recruited from the participants of the sample size. Weight was
measured by the electronic weighting machine. Body mass index was calculated by formula
that has weight in kg and height in meter square. BMI less than 25kg/m2 considered as
normal. After the confirmation of case and control by oral glucose tolerance test (OGTT), a
blood samples were collected in the volume of 6ml tubes. These samples were allowed to
freely clot, span 3000 rpm for 15 minutes by centrifuge machine. The serum collected by
centrifugation, collected in specific tubes and freez at -70oc. At the time of procedure, the
sample thawed by keeping them at the room temperature. The enzyme linked immune-
sorbent-assay(ELISA) was used to measure the cortisol and fluorescence immunoassay (FIA)
for the quantitative determination of HbA1c (hemoglobinA1c) in human whole blood.
Procedure of General ELISA:
The ELISA has been used as a diagnostic tool in medicine and plant pathology, as well as a
quality-control check in various industries. In simple terms, in ELISA, an unknown amount
of antigen is affixed to a surface, and then a specific antibody is applied over the surface so
that it can bind to the antigen. This antibody is linked to an enzyme, and, in the final step, a
substance containing the enzyme’s substrate is added. The subsequent reaction produces a
detectable signal, most commonly a color change in the substrate. Performing an ELISA
involves at least one antibody with specificity for a particular antigen. The sample with an
unknown amount of antigen is immobilized on a solid support (usually a polystyrene
microliterty plate) either non-specifically (via adsorption to the surface) or specifically (via
capture by another antibody specific to the same antigen, in a “sandwich” ELISA). After the
antigen is immobilized, the detection antibody is added, forming a complex with the antigen.
The detection antibody can be covalently linked to an enzyme, or can itself be detected by a
secondary antibody that is linked to an enzyme through bio-conjugation. Between each step,
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the plate is typically washed with a mild detergent solution to remove any proteins or
antibodies that are not specifically bound. After the final wash step, the plate is developed by
adding an enzymatic substrate to produce a visible signal, which indicates the quantity of
antigen in the sample.

Figure 3.1 generalized scheme of typical sandwich ELIZA protocol.

Estimation of cortisol:
Component used:
1. Precision pipettes to dispense 20, 50,100,150 and 300µl.
2. Disposable pipette tips.
3. Distilled or deionized water.
4. Plate shaker.
5. Micro well plate reader with a filter set at 450rm and an upper OD limit of 3.0 or
greater.
6. Cartridge Box
7. Cartridges
8. ID chip
9. Box containing detection buffer tubes
10. Detection Buffer Tubes i
 Procedure

30 μL (human serum/plasma/ control) or 50 μL (human whole blood) of sample was

transferred by using a transfer pipette to a tube containing the detection buffer.

Close the lid of the detection buffer tube and mixed the sample thoroughly by shaking
it about 10 times (sample mixed used immediately).

Pipette out 75 μL of a sample mixture and loaded it into the sample well on the
cartridge.

The sample loaded cartridge was inserted into the slot of the i-camber or a cartridge
before pushing it all the way inside the cartridge especially for this purpose.

Pressed the select button on the instrument for i-chroma test to start the scanning
process.

Instrument for i-chroma test would start scanning the sample loaded cartridge
immediately.

Read the test result on the display screen of the instrument for i-chroma test.

REAGENTS PROVIDED
1. Rabbit Anti-Cortisol Antibody Coated Micro Well Plate-Break Apart
Wells
Contents: One 96 well (12x8) polyclonal antibody-coated micro well plate in a
resalable pouch with desiccant.
Storage: Refrigerate at 2-8oC
Stability: 12 months or as indicated on label.

2. Cortisol-Horseradish Peroxidase (HRP) Conjugate Concentrate –

Contents: Cortisol-HRP conjugate in a protein-based buffer with a non-
mercury preservative. Volume: 300μl/vial
Storage: Refrigerate at 2-8oC
Stability: 12 months or as indicated on label.
Preparation: Dilute 1:100 in assay buffer before use (eg. 20 μl of HRP in 2 ml of
assay buffer). If the whole plate is to be used dilute 120 μl of HRP in 12ml of
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assay buffer. Discard any that is leftover.
 3. Cortisol Calibrators
Contents: Seven vials containing cortisol in a human serum-based buffer with a non-
mercury preservative. Prepared by spiking serum with a defined quantity of cortisol.
*Listed are approximate concentrations, please refer to vial labels for exact
concentrations. Storage: Refrigerate at 2-8oC
Stability: 12 months in unopened vials or as indicated on label. Once opened,
the standards should be used within 14 days or a liquated and stored frozen.
Avoid multiple freezing and thawing cycles.

Calibrator Concentration Volume/Via

l
Calibrator A 0 µg/dl 1.0 ml
Calibrator B 0.5 µg/dl 0.3 ml
Calibrator C 2 µg/dl 0.3 ml
Calibrator D 5 µg/dl 0.3 ml
Calibrator E 10 µg/dl 0.3 ml
Calibrator F 30 µg/dl 0.3 ml
Calibrator G 60 µg/dl 0.3 ml

Controls
Contents: Two vials containing cortisol in a human serum-based buffer with a
non-mercury preservative. Prepared by spiking serum with defined quantities
of cortisol. Refer to vial labels for the acceptable range.
Volume: 0.3 ml/vial
Storage: Refrigerate at 2-8 oC
Stability: 12 months in unopened vial or as indicated on label. Once opened,
the controls serum should be used within 14 days or a liquated and stored
frozen. Avoid multiple freezing and thawing cycles.
5. Wash Buffer Concentrate
Contents: One bottle containing buffer with a non-ionic detergent and a non-
mercury preservative.

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Volume: 50 ml/bottle
 Storage: Refrigerate at 2-8 oC
Stability: 12 months or as indicated on label.
Preparation: Dilute 1:10 in distilled water before use. If the whole plate is to be used
dilute 50 ml of the wash buffer concentrate in 450 ml of distilled water.
6. Assay Buffer
Contents: One vial containing a protein-based buffer with a non-mercury
preservative. Volume: 15 ml/vial
Storage: Refrigerate at 2-8oC
Stability: 12 months or as indicated on label.

7. TMB Substrate
Contents: One bottle containing tetra-methyl-benzidine and hydrogen peroxide
in a non-DMF or DMSO containing buffer.
Volume: 16 ml/bottle
Storage: Refrigerate at 2-8 oC
Stability: 12 months or as indicated on label.
8. Stop Solution
Contents: One vial containing 1M
sulfuric acid. Volume: 6 ml/vial
Storage: Refrigerate at 2-8 oC
Stability: 12 months or as indicated on label.
Procedure
1 working solutions of the cortisol-HRP conjugate was Prepared and washed with
buffer.
2 Remove the required number of micro-well strips. Reseal the bag and return any
unused strips to the refrigerator.
3 20 μl of each calibrator, control and specimen sample was added into correspondingly
labelled wells in duplicate.
4 100 μl of the conjugate working solution was added into each well (The use of a
multichannel pipette is recommended).
5 Incubate on a plate shaker (approximately 200 rpm) for 45 minutes at room
temperature.
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 6 Wash the wells 3 times with 300 μl of diluted wash buffer per well and tap the plate
firmly against absorbent paper to ensure that it is dry (The use of a washer is
recommended).
7 150 μl of TMB substrate was added into each well at timed intervals.
8 Incubate on a plate shaker for 15-20 minutes at room temperature (or until calibrator
A attains dark blue color for desired OD).
9 50 μl of stop solution was added into each well at the same timed intervals as in step7.
10 Read the plate on a micro-well plate reader at 450nm within 20 minutes after addition
of the stop solution.
11 *If the OD exceeds the upper limit of detection or if a 450nm filter is unavailable, a
405 or 415nm filter may be substituted. The optical densities will be lower, however,
this will not affect the results of the samples or control.

3.8 ELISA FOR HbA1c:

COMPONENTS
1 Micro ELISA plate
2 Lyophilized standard (1 vial or 2 vial)
3 Sample / standard dilution buffer (10ml/20ml)
4 Biotin-detection antibody (60ul/120ul)
5 Antibody dilution buffer (5ml/10ml)
6 HRP-Streptavidin conjugate (60ul/120ul)
7 SABC dilution buffer (5ml/10ml)
8 TMB substrate (5ml/10ml)
9 Stop solution(5ml/10ml)
10 Wash buffer (15ml/30ml)
11 Plate sealer (3/5 pieces)

MATERIAL REQUIRED
1. Microplate reader (wavelength: 450nm)
2. 37°C incubator
3. Automated plate washer
4. Precision single and multi-channel pipette and disposable tips
5. Clean tubes and Eppendorf tubes
6. Deionized or distilled water. i

REAGENT PREPRATION AND STORAGE

Bring all reagents to room temperature before use.
1: Wash Buffer: Dilute 30mL of Concentrated Wash Buffer was diluted into 750 mL of
Wash Buffer with deionized or distilled water. Put unused solution back at 4°C. If crystals
have formed in the concentrate, you can warm it with 40°C water bath (Heating temperature
should not exceed 50°C) and mix it gently until the crystals have completely dissolved. The
solution should be cooled to room temperature before use.

2: Standard:
 200ng/ml of standard solution: Add 1 ml of Sample / Standard dilution buffer into
one Standard tube, keep the tube at room temperature for 10 min and mix
thoroughly.
 100ng/ml→3.125ng/ml of standard solutions: Label 6 Eppendorf tubes were labeled
with 100ng/ml, 50ng/ml, 25ng/ml, 12.5ng/ml, 6.25ng/ml, 3.125ng/ml, respectively.
Aliquot 0.3 ml of the Sample / Standard dilution buffer into each tube. Add 0.3 ml of
the above 200ng/ml standard solution into 1st tube and mix thoroughly. Transfer 0.3
ml from 1st tube to 2nd tube and mix thoroughly. Then again 0.3 ml was
transformed from 2nd tube to 3rd tube and mix thoroughly, and so on.

3: Preparation of Biotin- detection Antibody working solution: prepare within 1 hour

before the experiment.
 Calculate the total volume of the working solution: 0.1 ml / well × quantity of wells.
(Allow 0.1-0.2 ml more than the total volume)
 The Biotin- detection antibody was diluted with Antibody dilution buffer at 1:100
and mix thoroughly. (i.e. Add 1 μl of Biotin- detection antibody into 99 μl of
Antibody dilution buffer.)

4: Preparation of HRP-Streptavidin Conjugate (SABC) working solution: prepare

within 30min before the experiment.
 Calculate the total volume of the working solution: 0.1 ml / well × quantity of wells.
(Allow 0.1-0.2 ml more than the total volume)
 SABC was Diluted with SABC dilution buffer at 1:100 and mix thoroughly. (i.e. Add
1 μl of SABC into 99 μl of SABC dilution buffer.)

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ASSAY PROCEDURE
1. Before adding standard sample and control (zero) wells the plate was wash 2 times,
2. 100μL standard or sample was added to each well for 90 minutes at 37°C.
3. 100μL Biotin- detection antibody working solution was added to each well
4. Aspirate and was washed 3 times
5. 100μL SABC working solution was added to each well. Incubate for 30 minutes at 37°C
6. Aspirate and washed it again 5 times
7. 90μL TMB substrate was added. Incubate 15 -30 minutes at 37°C
8.50μL Stop Solution was added. Read at 450nm immediately
9. Calculation of results for 60 minutes at 37°C.

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 RESULTS

4.0 RESULTS
In order to conduct the research, the subjects were recruited from (Jinnah hospital Lahore and
Sheikh zayed hospital Lahore) during march to august 2019. This case control study carried
out on a total 70 subjects.35 with diabetes and 35 without diabetes were selected. From them
those below age of 25 and above age of 50 patient samples were excluded. Thus 70 patients
(diabetic and control) are selected to see the interaction of cortisol secretion in type 2 diabetes
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patients of age 25-50.
Summarized data is given below in table 4.1

Table no 4.1 Total participants along with gender(Control

distribution
group) (Diseased group)

Male 70 35 50% 35 50%

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Other Physical parameters that observed in both groups (control &diseased)are body mass
index (BMI) and Age. In both groups age range is lie between 25 to 50 years.

Objectives
 To study cortisol level in diabetic and non-diabetic male patients.
 To study the levels of cortisol and glucose in male patients of diabetes mellitus type 2 of age
25-50.
 The study of interaction between cortisol secretion and male patients of type 2 diabetes
mellitus.

STATISTICAL ANALYSIS
The statistical analysis of abnormal cortisol level in patients with type 2 DM was confirmed
through compare the means by using one-way ANOVA. The data is collected for this
research was analyzed by using IBM SPSS version 23.for the discrete variables the data was
expressed in the form of mean and standard error of mean. Categorical data was expressed in
numbers (n) and percentage (%). Multiple statistical analyses were applied in order to find
out the results. The present study was conducted to determine the association of the four
variables that are age, BMI, HbA1c and cortisol with one another. At first reliability analysis
of the four scales i.e. ANOVA. Furthermore, Independent Sample t- test and Correlation
analysis were also computed.
The statistical analysis of abnormal cortisol levels in patients with type 2 DM was confirmed
through compare the means by using one way ANOVA.
The p value is significant less than 0.05, 0.001** is most significant while 0.000*** depicts
highly significant results. By using ANOVA, it was observed that parameters age (0.058) and
HbA1c (0.005) show significant results which means that with the increase of age production
of glucose in the body is disturbed in male patients. while on the other hand BMI (0.877) and
cortisol (0.208) show no significant results which indicates that they both have no effect on
each other as we see the interaction of cortisol and glucose these results shows that they both
independent. Cortisol secretion not depends on glucose. Glucose less and high amount not
affect the cortisol secretion. Shown in table 4.2 and figure no 4.1 and 4.2 as well.
By using t-test for the comparison of levels indicates that Age and HbA1c show significant
with the p value 0.038 which is smaller than 0.05
i (P≤0.05)HbA1c (glycatedhaemoglobin)
and cortisol show non-significant with the p value greater than 0.05.BMI and cortisol show
non-significant difference because both have p value greater than 0.05 (P≥0.05). The cohen’s
d less than .2 indicated that there is 0% of non-overlap in the control (control) and diabetic
male.as shown in table 4.3.
Figure 4.1 indicate that levels of cortisol show smaller difference between diabetic group and
non-diabetic group. Because they shows significant value is >0.5.
Figure 4.2 indicates the levels of HbA1c (glacated hemoglobin) is increased in diabetic
patients because they have significant value less than 0.05(P=≤0.05). So its shows that
HbA1c (glacated hemoglobin) in diabetic patients is more as compare to non-diabetic
patients. Figure 4.3 show somehow there is a relation between HbA1c and cortisol in diabetic
males. It’s not necessary that every diabetic male have higher level of cortisol but in some
cases diabetic males have also higher level of cortisol hormone. But in case of cushing
syndrome there is a compulsion that every diabetic patient have higher level of cortisol.
Figure 4.4 shows that levels of HbA1c abnormal in non-diabetic males. The levels of HbA1c
not affect the secretion of cortisol. It’s not necessary that HbA1c levels increased by the
increased secretion of cortisol.HbA1c and cortisol not depend to each other. Figure 4.5
indicate positive association between HbA1c cortisol secretion and BMI. In diabetic male
patients the value of HbA1c and cortisol high in diabetic persons.But the level of BMI same
as in diabetic and control group.

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Table 4.2: ANOVA:

Table 4.3: Independent Sample t-test to find out the differences in HbA1c, Cortisol and BMI
(N=70)

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i
Figure 4.1 levels of cortisol in(control and diabetic) groups are determined by
comparing their means by using one way ANOVA.

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Figure 4.2 levels of HbA1c (glacated hemoglobin) in (control and diabetic) groups are
determined by comparing their means by using one way ANOVA.

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 Fi
gure 4.3 levels of HbA1c (glacated hemoglobin)and cortisol in diabetic patients determined
by comparing their means by using one way ANOVA.

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 Figure 4.4 levels of HbA1c (glacated hemoglobin) in (control and diabetic)patients
determined by comparing their means by using one way ANOVA.

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Figure 4.5 associations of BMI HbA1c and cortisol in diabetic male and non-diabetic male.

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DISCUSSION

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5.0 DISCUSSION
Cortisol affects various parts of our body, both psychologically and physically. Some
ailments caused by increased cortisol levels include a suppressed immune system, insomnia,
severe mood swings, depression and severe hypotension. Glucocorticoids inhibit
inflammatory response; specifically, cortisol suppresses the synthesis and secretion of
arachnidonic acid, a key precursor for a number of mediators of inflammation. However,
heightened levels of glucocorticoid hormones can lead to suppression of the body’s immune
response due to stabilization of lysosomes, decrease in number of circulating T4 lymphocytes
and a decrease in production of key mediators in immune Cortisol can help control blood
sugar levels, regulate metabolism, help reduce inflammation, and assist with memory
formulation. It has a controlling effect on salt and water balance and helps control blood
pressure. Diabetes is a set of metabolic illnesses characterized via hyperglycemia attributable
to defects in insulin secretion, insulin movement, or each. The persistent hyperglycemia of
diabetes is associated with lengthy-time period damage, dysfunction, and failure of various
organs, specifically the eyes, kidneys, nerves, coronary heart, and blood vessels. Numerous
pathogenic strategies are concerned within the development of diabetes.. The deficiency of
insulin in target tissues provides basis for carbohydrate, fat and protein metabolism abnormalities in
diabetes. Poor Insulin action results inadequate secretion or decreased tissue response to insulin at one
or more junctions in the complex pathway. The prime cause of hyperglycemia is impairment of
insulin secretion and insulin action defects often coexists in the same patient.  Age is an important
factor, the frequency of diabetes mellitus increases with age. Impaired renal function and increased
sympathetic nervous system activity and post receptor impairment of fat tissue occurs due to Glucose
mediated insulin secretion decreases and glucose tolerance impairment. However, in different studies,
it has been observed that cortisol secretion does not change by age rather a decrease has been
observed in its catabolic reactions. This type of diabetes is caused by relative insulin deficiency and
insulin remissness to perform its task. Individuals suffering from Type 2 diabetes patients are mostly
overweight because in response to increased fat content in the body, insulin resistance
develops(Arora, Ojha et al. 2009).A number of genetic and environmental factors interact to produce
a heterogeneous and progressive disorder with variant degrees and levels of insulin resistance and
pancreatic βcell dysfunction. When β-cells are no longer able to secrete enough insulin to overcome
insulin resistance, impaired glucose tolerance progresses to type 2 diabetes Insulin resistance and
impaired glucose tolerance more oftenly results due to overweight and obesity.
Although the specific etiologies are not known, autoimmune destruction of b-cells does not
occur, and patients do not have any of the other
i causes of diabetes listed above or below.
Most patients with this form of diabetes are obese, and obesity itself causes some degree of
insulin resistance. Patients who are not obese by traditional weight criteria may have an
increased percentage of body fat distributed predominantly in the abdominal region.The
current study proclaimed that age showed significant results with diabetes that referred there
is link between age and diabetes. Old aged individuals had more chances of developing the
disease than adult ones. However, a study conducted by Connor and colleagues revealed that
individuals aged between 45–64 years and those 65 and older had better glycemic control and
perceived less adverse impacts of diabetes on their quality of life as compared to younger
patients. More chances of developing the disease among Younger adults are related to
effective and aggressive care, and accessed care less frequently (Connor et al., 2003).The
present study conferred that occurrence of diabetes depends upon secretion of HBA1C that
showed significant results with diabetes. One of the previous research proposed that Serum
ferritin was significantly higher in diabetic patients when compared to controls and serum
ferritin had a positive correlation with prolong duration of diabetes. There was a positive
correlation between serum ferritin and HbA1c. This indirect relation showed that increased
incidence of diabetes is related to more secretion of serum ferritin and thus, HbA1c (Raj and
Rajan, 2013). Another study demonstrated HbA1c as an effective screening tool for detection
of Type 2 diabetes. HbA1c has less intra individual differences and better predicts both micro
as well as macro vascular problems (Bennett et al., 2007). The current study showed that
BMI had no significant relationship with occurrence and prevalence of diabetes. A study
carried out by Looker and co-workers depicted that weight loss was generally seen and mean
rate of change of BMI ranged between 0.61 and 0.22 kg/m 2 per year among diabetic patients.
Before advancement of diabetes, there was a progressive rise in weight, and after diagnosis,
there was an inclination toward weight loss (Looker et al., 2001).In this study it was
evaluated that secretion of cortisol had no association with occurrence of diabetes. Previous
studies had yielded conflicting results on association between type 2 diabetes status and
cortisol topographies. Similar to the findings of present research workVreeburg and his co-
researchers found no association between type 2 diabetes and diurnal cortisol slope.
Conversely, two different studies conducted by Lederbogen and Hackettreportedan
association between type 2 diabetes status and flatter daily cortisol levels, decline across the
day and higher bedtime cortisol levels even in their fully adjusted model.

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 SUMMARY

6.0 SUMMARY
Diabetes mellitus is one of the serious health concerns thought the whole world. The
occurrence and severity of disease depends upon secretion of many hormones, cortisol is one
of them. In this study, effect of cortisol secretion is evaluated in 35 diabetic and 35 non-
diabetic subjects. It was assessed on the basisi of physical i.e. BMI and age, as well as
biochemical parameters i.e. HbA1c associated with cortisol secretion. It was noticed that
BMI has no effect on occurrence of diabetes whereas old age individual were observed to be
at high risk of diabetes mellitus. This study contradicted previous research work carried out
to assess cortisol concentrations in diabetic patients. It referred that cortisol secretion has no
association with prevalence of disease rather it was find out to be an age dependent disorder.

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CONCLUSION

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7.0 CONCLUSION

 Type 2 diabetes mellitus is a result of interaction between genetic and environment

factors, leading to heterogeneous and progressive pancreatic β-cell dysfunction.

 The inability of β cells to secrete enough insulin produces type 2 diabetes.

Abnormalities in other hormones secretion of some hormones increase and decreased
which affects the human immune system.

 In conclusion, this study has found abnormally decreased the level of cortisol with the
passage of time in type 2 diabetic male subjects. Moreover, there is no specific link of
cortisol with type to diabetes mellitus.

 Age and HbA1c showed significant results which means that with the passage of time
production of glucose in the body is disturbed in male patients

 BMI show no significant results with cortisol which indicates that they both have no
effect on each other they are both independent.

 The prevalence of diabetes mellitus type 2 is less in male of age 25 to 35.But the
prevalence of diabetes mellitus type 2 is high in male of age 35 to50.