1.

INTRODUCTION

Insulin resistance is a feature of metabolic syndrome. It is strongly associated with

dyslipidaemia, obesity, non-alcoholic fatty liver disease (NAFLD) and hyperglycaemia, and
type 2 diabetes mellitus (T2DM). Hepatic insulin resistance is a key factor related to many
obesity-associated pathophysiological conditions such as diabetes and NAFLD (Liping Gu et
al.,2019). Excessive caloric intake, either from a high-fat diet (HFD) or sugar-sweetened
bevarages (SSB), is associated with development of hepatic insulin resistance. In T2DM, HGP
is higher in the post-absorptive state and fails to be properly suppressed by insulin, which
results in primarily from excessive gluconeogenesis rather than glycogenolysis (Samir softic
et al.,2020).

Glucose is an indispensable fuel for all cells and organs, but at the same time leads to
problems at high concentrations. As a consequence, blood glucose is controlled in a narrow
range to guarantee constant supply and on the other hand avoid damages associated with
elevated glucose levels. (DeFronzo RA et al.,2004). There is a general consensus that in the
postabsorptive state, the liver is the main source of free glucose generated via glycogenolysis
and gluconeogenesis (Stephen F. Previs et al,.2009).

The liver is crucial for the maintenance of normal glucose homeostasis and it produces
glucose during fasting and stores glucose postprandially. However, these hepatic processes are
dysregulated in type 1 and type 2 diabetes mellitus, and this imbalance contributes to
hyperglycaemia in the fasted and postprandial states. Net hepatic glucose production is the
summation of glucose fluxes from gluconeogenesis, glycogenolysis, glycogen synthesis,
glycolysis and other pathways (Max C. Petersen et al., 2018).

Liver is the main organ controlling blood glucose by (i) releasing newly synthesized or
stored glucose in the blood stream when blood glucose is low (ii) using and storing glucose
when blood glucose is elevated (Konig et al.,2012). In the liver, defects in insulin-stimulated
hepatic glycogen synthesis and increased rates of hepatic gluconeogenesis are the main factors
that contribute to insulin resistance and fasting hyperglycemia (Erion and Shulman 2010).
Chronic hyperglycemia of diabetes is associated with long-term damage, dysfunction, and
failure of different organs. The vast majority of cases of diabetes fall into two broad
etiopathogenetic categories: type-1 and type-2 diabetes (Fatima Z Syed et al.,2022)
 Gluconeogenesis is an endogenous metabolic pathway in which non-carbohydrate
substances are converted to glucose. This process is essential for maintaining blood glucose
during starvation, long-term muscle work, and when the body consumes more fats and proteins
than carbohydrates and contributes to increased glycemia in diabetes mellitus as well as
disorders associated with insulin resistance and muscle wasting. (Buchanan et al.,1999). The
major substrates for gluconeogenesis are lactate, glycerol, and gluconeogenic amino acids. The
main source of lactate is anaerobic glycolysis of red blood cells, the renal medulla, and skeletal
muscle. Therefore, lactate only contributes to glucose recycling, whereas other substrates
provide a new carbon skeleton (Milan Holecek et al,.2023) .

Hepatic gluconeogenesis are essential processes for the prevention of hypoglycemia

during short-term starvation. production is regulated by the interaction of different regulatory
mechanisms, by glucoregulatory hormones, glucose itself, and gluconeogenic substrates. In the
last decades, more insight has been gained into the importance of the autonomous nervous
system and the existence of an extensive paracrine network in the liver that seems to exert a
potent glucoregulatory role as well. Maintainance of a constant blood glucose level is essential
for normal physiology in the body, particularly for the central nervous system. The brain can
neither synthesize nor store the amount of glucose required for normal cellular function.

In the postabsorptive state, glucose is the obligatory fuel for the brain and provides
more than 90% of the energy needed for brain function. After absorption of the last meal,
endogenous glucose production gradually increases to prevent hypoglycemia. Liver glycogen
stores, as measured by liver biopsy, are limited to approximately 70 to 150 g after an overnight
fast; the kidney does not store glycogen. Consequently, glucose production during more
prolonged starvation depends on hepatic and renal gluconeogenesis.

The classical hormones involved in the regulation of hepatic glucose production are
insulin and the counterregulatory hormones glucagon, epinephrine, norepinephrine, cortisol,
and growth hormone. Besides their specific effects, all glucostimulatory hormones act by
increasing cytosolic free calcium levels, either directly or by stimulation of IP3 synthesis.
Increasing calcium levels is sufficient by itself to stimulate gluconeogenesis. (E.P.M. Corssmit
et al.,2001).
Insulin

Shortly after the discovery of insulin, it was shown that insulin inhibits hepatic glucose
production and stimulates peripheral glucose uptake. After an overnight fast, insulin
concentrations decrease to basal levels (5 U/mL, equivalent to (35 pmol/L). This results in a
major decrease in glucose uptake by insulin-dependent tissues such as resting muscle and
adipose tissue, which can use FFAs for their energy supply instead of glucose. Consequently,
glucose is available for non-insulin-dependent tissues such as the brain, blood cells, and renal
medulla, which depend strongly on glucose for their energy supply (Salim Bastaki et al., 2005).

Insulin inhibits gluconeogenesis by inhibiting the transcription of the gene of one of the
main gluconeogenic enzymes, phosphoenolpyruvate carboxykinase (which converts
oxaloacetate into 2-phosphoenolpyruvate) and by increasing the transcription of the gene
(Anila K Madiraju et al.,2019). Glycogenolysis seems to be more sensitive than
gluconeogenesis to inhibition by small increments in insulin secretion. The decrease in plasma
insulin from postprandial to basal concentrations is crucial for the stimulation of hepatic
glycogenolysis (Guoqing cao et al.,)

The effects of insulin on hepatic gluconeogenesis are less potent, are more complex,
and occur through multiple mechanisms. The direct inhibitory effect of insulin on the
transcription and activity of key hepatic gluconeogenic enzymes through forkhead box class
O-1 (FOXO1) phosphorylation, including phosphoenolpyruvate carboxykinase (PEPCK) is
well established. Further, insulin inhibits the secretion of glucagon, a known activator of
gluconeogenesis, thereby bringing about an indirect inhibitory effect on the process in the liver.
(Dale S. Edgerton et al.,2009)
 2. REVIEW OF LITERATURE

Diabetes mellitus (DM) is a metabolic disease caused by a malfunction in either the action
or secretion of insulin, or both. In turn, a lack of insulin causes chronic hyperglycemia along
with abnormalities in the metabolism of proteins, fats, and carbohydrates. The most prevalent
endocrine condition, diabetes mellitus is predicted to affect over 200 million individuals
globally by 2010, with another 300 million expected to develop the condition by 2025. As the
condition worsens, tissue or vascular damage occurs, which can result in serious diabetes
consequences such ulceration, retinopathy, neuropathy, nephropathy, and cardiovascular
problems. (Salim Bataki ,2005)

Classification of diabetes

 Type 1 diabetes (insulin deficit)

 Type 2 diabetes (insulin resistance)

are the two main forms of diabetes mellitus. Diabetes is defined as a hyperglycemic
state either fasting or postprandial. According to the International Diabetes Federation (IDF)
the overall prevalence of diabetes mellitus was 366 million in 2011 and is expected to rise to
552 million by 2038. (Whiting DR et al. 2011)

 Fasting blood glucose ≥ 126 mg/dl.

 Random blood glucose ≥ 200 mg/dl

The ADA includes haemoglobin (HbA1C), which is a marker of hyperglycemia management.

A concentration of 248 mmol/mol. Prediabetes is defined as a blood glucose level of between
100 and 125 mg/dl, or 5.6 to 6 g mmol/L, after an overnight fast. Several lifestyle studies have
shown that increasing physical activity leads to a 5-10% weight loss. Diabetes type 2 has a high
risk of cardiovascular disease. American Diabetes Association (2010a) and American Diabetes
Association (2010b).

The classification often depends on the clinical presentation at diagnosis and it is

common clinical practice to classify individuals based on the following variables:

1. age at onset of diabetes

2. the abruptness of hyperglycemia

3. presence of ketosis at presentation

4. degree of obesity

5. need for insulin at diagnosis

Type 1 and 2 have a genetic propensity and environmental influences that influence
how the genetic tendency manifests itself. The main problem in type 1 diabetes is a widespread
lack of beta cell activity. Insulin resistance arises in type 2 diabetes, making it impossible to
attain or sustain the significant compensatory increases in insulin secretion required to preserve
normal glucose tolerance. The patient moves from normal glucose tolerance to impaired
glucose tolerance, diabetes with mostly postprandial hyperglycemia, and finally diabetes with
fasting hyperglycemia as beta cell function continues to decline. (Lebovitz, 2001).

Type 1 diabetes mellitus

The pancreas is unable to produce insulin due to the destruction of the beta-cells that
can produce insulin hormone. It affects about 3–5% of people with diabetes and generally
occurs in childhood or adolescence but can also occur in adults. Type 1 diabetes is therefore
characterized by beta-cell destruction, on an autoimmune or idiopathic basis, which leads to
absolute insulin deficiency. (Tiziana Ciarambino et al.,2022). . Insulin replacement is essential
for management because this leads to hyperglycemia and ketosis. People with T1DM live for
a long time, prevalence is highest in adults. Polyuria, polydipsia, and weight loss are among
the symptoms. Diabetic ketoacidosis is one of the acute consequences that needs to be treated
right away. Both macrovascular and microvascular diseases are long-term consequences.
(Fatima Z Syed,2022).

Type 2 diabetes mellitus

Type 2 diabetes, also known as non-insulin-dependent diabetes, is the predominant type

of diabetes, constituting approximately 90-95% of all cases of diabetes. Insulin resistance or
insufficient insulin production in the body leads to the inability to maintain normal blood
glucose levels. Type 2 diabetes is frequently linked to lifestyle factors such as obesity,
sedentary behavior, and an unhealthy diet. The prevalence of this condition is predominantly
observed in adults; however, there has been a notable increase in the diagnosis of children and
adolescents, which can be attributed to the growing issue of childhood obesity. (FNU Suganth
et al.,2023)
Gestational diabetes

Pregnancy-related hyperglycemia increases the risk of bad outcomes for the mother,
fetus, and newborn. This risk is present whether the hyperglycemia adopts the T2D form
diagnosed before or during pregnancy. Newborns born to mothers with gestational diabetes are
at an elevated risk of developing diabetes in adulthood. The increased incidence of pregnancy-
related complications, such as premature birth, large-for-gestational-age births, macrosomia
(birth weight > 4.5 kg), cesarean delivery, and preeclampsia is primarily due to hyperglycemia
during pregnancy, which leads to larger neonates. Gestational diabetes can be influenced by
several risk factors, such as having a family history of the condition, being obese, advanced
maternal age, having polycystic ovarian syndrome, leading a sedentary lifestyle, and exposure
to environmental pollutants. The identification of gestational diabetes relies on specific criteria,
which involve evaluating fasting blood sugar levels, blood sugar levels after a 75 g oral glucose
load, and other relevant parameters. (Uazman alam et al.,2013).

Hybrid forms of diabetes

Slowly evolving immune-mediated diabetes, also known as latent autoimmune diabetes

in adults (LADA) resembles type 2 diabetes clinically but is characterized by the presence of
pancreatic autoantibodies associated with autoimmune diabetes. Initially, individuals with
LADA can be managed with oral medications and lifestyle modifications like those with type
2 diabetes. However, they tend to progress to requiring insulin therapy at a faster rate compared
to typical type 2 diabetes patients. There is a comparable subtype known as latent autoimmune
diabetes in youth (LADY) observed in children and adolescents with clinical type 2 diabetes
and pancreatic autoantibodies. The criteria used to diagnose LADA typically involve positive
glutamic acid decarboxylase (GAD) autoantibodies, age older than 35 years at the time of
diagnosis, and no immediate need for insulin therapy in the first 6–12 months after diagnosis.
The prevalence of GAD autoantibodies in clinically diagnosed type 2 diabetes individuals
varies between ethnic and regional groups, ranging from 5% to 14%. (Samar A. Antar et
al.,2023).
Antioxidants:

Today, antioxidants are an essential component of our life because they neutralize or
eliminate free radicals, also known as "reactive oxygen species" (ROS), before they cause cell
harm. The disintegration of cell membranes, damage to membrane proteins, and DNA
mutations brought on by ROS-induced oxidation cause aging and further trigger or spread the
onset of numerous illnesses, including cancer, diabetes mellitus, arteriosclerosis, liver damage,
inflammation, skin damage, coronary heart disease, and arthritis. (Sunitha Dontha,2016)

There are several degrees of cellular defense against oxidative stress, which is the
harmful impact of reactive oxidants produced during aerobic metabolism. Three degrees of
protection are included in defense strategies: repair, interception, and prevention. The
localization of antioxidant chemicals and enzymes as well as the maintenance of sufficient
antioxidant levels are all part of the regulation of antioxidant capacity. Cell specialization in
these tasks and adaptation in the short and long term are fresh topics of investigation. It is
essential to regulate the activity of prooxidant enzymes like NO synthases and NADPH
oxidase. Artificial antioxidants imitate biological processes. (Britton,1993).

An antioxidant is a chemical capable of mitigating the free radicals in the human

defense system. These are the substances that suppress the oxidation of any molecules, when
present in very low concentrations. Antioxidants neutralize the detrimental effects of free
radicals produced as a result of metabolism (Young IS et al., 2001), Various bioactive
antioxidants are seen in medicinal plants. Enzymatic and non-enzymatic antioxidants are the
two types of antioxidant molecules (Ramasarma T et al., 2007)

A) Enzymatic antioxidant:
 Catalase
 Glutathione peroxidase
 Glutathione reductase
 Superoxide dismutase

Catalase is an antioxidant enzyme that converts hydrogen peroxide to oxygen and water which
counteracts the effect of hydrogen peroxide found within cells. Most catalase activity during
tissue manipulation are destroyed. An imbalance between reactive oxygen species and
antioxidant interaction causes oxidative stress. A lot of diseases have oxidative stress as an
etiologic or exacerbating component (Aly DG et al., 2010).
Superoxide dismutase catalyzes the dismutation of superoxide and is found throughout the
body. As a side effect of this reaction, hydrogen peroxide is formed, which aids in the
transmission of free radical harm. Hydrogen peroxide, superoxide, and hydroxyl radicals are
only a few of the reactive oxidants produced by the human body (Ramasarma T, et al., 2007).

Antioxidant enzymes such as glutathione peroxidase and glutathione reductase were

found in the body. Reduced Glutathione in its reduced form has a protective function. The
oxidized form does not offer any protection. Reduced glutathione aids in the neutralization of
hydrogen peroxide generated within the cell. These enzymes serve a crucial role in reducing
oxidative stress levels. Glutathione is a free radical scavenger due to its frequent oxidation and
reduction. (Champe PC et al.,2004).

B) Non-enzymatic antioxidants:

 Vitamin A, C, E
 Polyphenols
 Phenols
 Flavonoids

Polyphenols are polyhydroxylated phytochemicals produced by plants that offer a

variety of health benefits. Polyphenols have the ability to scavenge and trap free radicals by
donating hydrogen ions to stabilize them. (Hollman P.C.H et al., 2005).

Phenolic acids are benzoic and cinnamic acids that have been hydroxylated. In foods,
hydro benzoic acid is typically found as glucosides, but hydro cinnamic acid such as p-
coumaric, caffeic, and ferulic acid, is mostly found as simple esters (Mattila P et al.,2002).

Flavonoids are a vast group of polyphenolic chemicals with a benzo-y-pyrone structure

that are extensively found in plants. The antioxidant activity of flavonoids is their most
important function. By quenching singlet oxygen, flavonoids can scavenge numerous oxidizing
species such as superoxide anion, hydroxyl, and peroxyl radicals (Harborne JB et al., 2000).

Ascorbic acid or vitamin C is an organic acid that is the most crucial vitamin for human
nutrition. Ascorbic acid is synthesized in mitochondria and transported to other cell through
proton-electron chemical gradient or facilitated diffusion. It also has the ability to scavenge
many types of free radicals. The main sources of ascorbic acid are fruits and vegetables.
Ascorbic acid can fight against chronic diseases, such as cardiovascular diseases and certain
types of cancer (Ohad I et al., 2003).
 3. AIM AND OBJECTIVE

The present study entitled “Evaluation of Anti diabetic effect of insulin on fructose induced
gluconeogenesis in goat liver” were designed with following objectives,

 To investigate hyperglycemic condition in goat liver tissue when exposed to fructose.

 To study the role of insulin in regulating fructose-induced gluconeogenic activity.

 To assess the enzymatic activity of key gluconeogenic enzymes (e.g.,

phosphoenolpyruvate carboxykinase, fructose-1,6-bisphosphatase) under the insulin
treatment.

 To compare the metabolic response of goat liver to insulin in the presence and absence
of fructose.

 To assess the influence of fructose and insulin on hepatic glycogen content to determine
how these factors affect glycogen storage and mobilization in goat liver tissues.
 4. MATERIALS AND METHODS

Phase I

 Tissue preparation
 Manual homogenization
 Centrifugation
 Extracting supernatant

Experimental Setup

 Group I : Goat liver tissue

 Group II : Induction of Fructose(20mM)
 Group III: Induction of Fructose and Treatment of Insulin (5nM)

Phase II

Estimation of altered metabolites

 Glucose
 Fructose
 Carbohydrates
 Protein
 Free amino acids
 Pyruvate

Phase III

Enzyme assays

 Alanine transaminase
 Aspartate transaminase
 Lactate dehydrogenase

Phase IV

Estimation of enzymatic assay

 Catalase activity
 Total antioxidants
Estimation of Non-Enzymatic assay

 Estimation of Ascorbic acid

PHASE V

 Glucose uptake assay

 Methionine uptake assay
 Tryptophan uptake assay
 Succinate dehydrogenase
PHASE II

ESTIMATION OF GLUCOSE BY ORTHO TOLUIDINE METHOD

(Trinder,1998)

Principle

Glucose react with ortho-toluidine to give N-glucosamine and treated with glacial acetic
acid in which it gives green colour and colour developed is read calorimetrically at 640nm.

Reagents: Appendix I

Procedure

Into the series of test tubes working standard glucose solution was pipetted out in a
range of concentration 20-100µg and total volume was made upto 1ml with distilled water to
all the tubes .4ml of ortho-toluidine reagent added to all the tubes. Heat all the tubes in boiling
water bath for 10 minutes, cooled and read at 640nm. Made a standard graph with concentration
of the standard on the X-axis against its absorbance on Y-axis.

ESTIMATION OF FRUCTOSE BY SELIWANOFF’S METHOD

(Krishveni et al.,1984)

PRINCIPLE

Fructose in the presence of acid dehydratase to give a furfural derivative which form
complex with resorcinol to give a red colour complex which can be determined calorimetrically
at 520nm.

Reagents: Appendix II

Procedure

A working standard comparable to 40 -200 μg added to series of test tubes with distilled
water the capacity in all of the test tubes were increased to 2 ml. In each tube,4 ml of seliwanoff
reagent is added to all the tubes and boiled for 10 minutes. The test tubes cooled at room
temperature. The intensity of color developed was measured at 520nm. Concentration was
plotted on the X-axis and Y-axis of a typical graph. From the graph the concentration of the
unknown was determined.

ESTMATION OF CARBOHYDRATES BY THE ANTHRONE METHOD

(F.W. Yemm and A.J. Willis et al., 1954)

Principle

Using weak hydrochloric acid, carbohydrates are first hydrolyzed into simple sugars.
Glucose is dehydrated to hydroxymethyl furfural in a hot acidic media. This compound reacts
with anthrone to produce a green-colored product with a maximum absorption wavelength of
630nm.

Reagents: Appendix III

Procedure

0.2, 0.4, 0.6, 0.8, 1.0 ml pipetted as the working standard along with blank and make
up the volume to 1 ml. Then 5 mL of anthrone reagent is added and heat for 8 minutes, cooled
and read at 630nm. Make a standard graph with concentration of the standard on the X-axis
against its absorbance on the Y-axis.

ESTIMATION OF TOTAL PROTEIN (Lowry et al., 1951)

Principle

In an alkaline medium, the aromatic amino acids tryptophan, tyrosine and peptide bond
create a complex with the cupric ion reducing the phenol reagent containing phosphomolybdic
acid and tungstic acid to a molybdenum blue colored complex which is measured
colorimetrically at 670nm.

Reagents: Appendix IV

Procedure

Working standard protein concentration in the range of 40-200µg of protein were

pipetted out into a series of test tubes and the final volume in all the tubes was made up to 1 ml
with distilled water. A blank unknown protein solution of an adequate volume was also
produced up to 1 ml with distilled water. 5 ml of protein reagent was added to each tube, mixed
thoroughly and allowed to stand for 20 minutes. After that 0.5 ml of the phenol reagent were
added to each test tube and mixed thoroughly. After 20 minutes incubation the generated blue
colored was measured at 670nm against the reagent blank.

ESTIMATION OF FREE AMINO ACID (Tiwari & et al. 2015)

Principle

Ninhydrin, a potent oxidizing agent, decarboxylates the ∝-amino acid to produce a

highly bluish-purple product that can be measured at 570 nm using calorimetrically.

Reagents: Appendix V

Procedure

A working standard comparable to 20 -100 μg added to series of test tubes with distilled
water the capacity in all of the test tubes were increased to 1 ml. In each tube,1 ml of ninhydrin
was added except the blank that lacked the amino acid glycine. All the tubes were heated for
20 minutes and 5 ml of diluent solution added and thoroughly mixed. The intensity of purple
color developed was measured at 570 nm after 15 minutes and this color remains stable for an
hour. Concentration was plotted on the X-axis and Y-axis of a typical graph. From the graph
the concentration of the unknown was determined.

ESTIMATION OF PYRUVATE ([Link] al., 1961)

Principle

In the presence of sodium hydroxide, pyruvic acid produces a color with dinitrophenyl
hydrazine that may be read calorimetrically at 540nm.

Reagents: Appendix VI

Procedure

In separate test tubes, pipetted out 10-50 µg of standard pyruvic acid solution and 0.1
ml of the unknown sample solution and made up the volume to 3 ml with distilled water.1 ml
of DNPH reagent was added to all the test tubes and incubate for 20 minutes and add 2ml of
1N NaOH then incubate for 20 minutes at 540nm the color developed is read at
calorimetrically.
PHASE III

ASSAY OF ENZYME ALANINE TRANSAMINASE (Milton H Saifer et

al.,1966)

Principle

The enzyme is allowed to act on the substrate and the pyruvate is permitted to react
with 2,4-DNPH and at 520nm the color developed was read.

Reagents: Appendix VII

Procedure

1 ml of substrate was pipetted out into tubes test and control. Incubate the tubes for 10
minutes at 37°C and add 0.2 ml of supernatant followed by incubation for 30 minutes before
adding 1 ml of 2,4-DNPH to both of test and control. After that 0.2 ml of supernatant was added
to control and the developed color was measured at 520 nm. Pipette 0.2-1.0 ml of standard into
a succession of test tubes increasing the final volume to 5 ml with buffer.1 ml of 2,4-DNPH
was poured to each test tubes and incubate at 37°C for 30 minutes before adding 2 ml of IN
NaOH and the color developed was read at 520 mm.

ASSAY OF ENZYME ASPARTATE TRANSAMINASE (Xing-Jiu Huang

et al.,2006)

Principle

The enzyme is allowed to work on the substrate and the liberated oxaloacetate is
allowed to react with 2,4-DNPH with the color generated being measured at 520 nm and
compared to a standard.

Reagents: Appendix VIII

Procedure

2 ml of substrate pipetted into test and control tubes and incubate for 10 minutes at
37°C. Incubate for 1 hour after adding 0.2 ml of supernatant as the test. The addition of 1 ml
of 2,4-DNPH to both test and control is done. Finally, the control tube receive 0.2 ml of
supernatant and the developed color was measured at 520 nm the color intensity.
 0.2-1.0 ml of standard pipetted into a succession of test tubes increasing the final
volume to 5 ml of buffer. 1 ml of 2,4-DNPH was added and incubated at 37°C for 30 minutes
before adding 2 ml of 1N NaOH and the color obtained was read at 520 nm.

4.2.3. ACTIVITY OF ENZYME LACTATE DEHYDROGENASE (Briere

et al., 1966)

Principle

Lactate dehydrogenase reacts with the lactate to produce pyruvate. With 2,4-DNPH
Pyruvate produces phenyl hydrazine. At 520 nm the color developed was measured.

Reagents: Appendix IX

Procedure

In each test and control tube pipette out 0.6 ml of buffered substrate reach. To test tubes,
add 0.2 ml of demineralized water mix well and incubate at 37°C for 15 minutes.0.2 ml NAD+
was added to each tube and thoroughly mixed. Tubes were incubated at 37 °C for 15 minutes
0.2 ml was added to the test and control tubes and thoroughly mixed. The reaction was arrested
by 1 ml of DNPH mix well and incubate for 15 minutes at 37°C. To this 2ml 1N NaOH was
added and the color was read at 540 mm. The standard is performed by adding all the reagents
as given in the protocol.

4.3. PHASE IV

4.3.1. TOTAL ANTIOXIDANT ASSAY (Prieto et al., 1999)

Principle

The reduction of phosphomolybdic acid to phosphomolybdenum blue complex by

sodium sulfide is the basis for this approach. The phosphomolybdenum blue complex produced
is oxidized by the addition of nitrite resulting in a reduction in the blue color intensity.

Reagents: Appendix X

Procedure

The phosphomolybdenum antioxidant assay method used to determine the total

antioxidant activity. In a test tube 1 ml of reagent solution containing 0.6M sulphuric acid,
28mM sodium phosphate and 4mM anamonium molybdate was coupled with 0.1-0.5 ml of the
chemical dissolved in water. The tubes were capped and incubated for 90 minutes at 95°C in a
thermal block. The absorbance of each tube's solution measured at 695nm against a blank after
cooling to room temperature. Standard ascorbic acid (100-500 µg/ml) was used in the same
way. The quantity of ascorbic acid was used to calculate the antioxidant activity.

ENZYMATIC ANTIOXIDANT

4.3.2. ASSAY FOR CATALASE (Barber JM et al., 1980)

Principle

When dichromate in acetic acid was heated in the presence of hydrogen peroxide was
transformed to perchloric acid and then to chromic acetate. Spectrometrically, the chronic
acetate generated was measured at 620nm. The addition of dichromate acetic acid combination
stops the reaction at a predetermined time interval and the remaining hydrogen peroxide is
detected by measuring chromic acetate.

Reagents: Appendix XI

Procedure

The sample (0.1g) with 0.1M phosphate buffer, pH 7.0 in a prechilled mortar and pestle.
Centrifuged at 10,000 rpm at 4·c for 10 minutes. The supernatant used as enzyme extract. 1ml
of enzyme extract taken in three different conical flask. Add 3ml of phosphate buffer, 2ml of
hydrogen peroxide in all the conical flask. 10ml of sulphuric acid is added and titrate against
potassium permanganate until a faint purple colour persist for 15 seconds.

ASSAY OF SUPEROXIDE DISMUTASE (SOD) (Christine J. Weydert et

al.,2010)

Principle

By monitoring the rate of color generated at 460 nm, the rate of breakdown of hydrogen
peroxide by superoxide dismutase with Ortho dianisidine as a donor can be assessed.

Reagents: Appendix XII

Procedure

0.2 mL of the sample weighed and mixed with 2 ml sodium phosphate buffer, centrifuge
for 5 minutes at 2000 rpm, and use the supernatant for the test. To 0.2 ml of enzyme extract,
0.06 ml of ortho-dianisidine and 0.2 ml of riboflavin were added. With phosphate buffer, the
final volume was increased to 5 ml. The test mixture was exposed to sunlight or UV for 15
minutes and read at 400 nm within 30 minutes. Simultaneously with the sample, a control
without the enzyme mixture was run.

NON ENZYMATIC ANTIOXIDANT

ESTIMATION OF ASCORBIC ACID (Sadasivam and Theymoli,1987)

Principle

The 2,4-dichlorophenol indophenol dye was reduced to a colourless leuco-base by

ascorbic acid. Dehydro ascorbic acid was formed when ascorbic was oxidized. Despite the fact
that the dye is a multi-coloured the result is a pink color. In an acidic media the dye is pink in
color. As a titrating medium oxalic acid was utilized.

Reagents: Appendix XIII

PROCEDURE

5 ml of the working standard solution pipetted into a conical flask with a capacity of
100ml. Titrate against the color with 10ml of 4% oxalic acid (V1 ml). The arrival of pink color
that lasts a few minutes is the end point. The amount of colour is the same as the amount of
ascorbic acid swallowed. In 4% oxalic acid extract the sample and dilute to known volume
(100ml) before centrifuge. Pipette off 5ml of the supernatant mix in 10ml of 4% oxalic acid
and titrate against the dye (V2 ml).

PHASE V

GLUCOSE UPTAKE ASSAY (CirilloV.P et al., 1962)

Principle

The rate of glucose uptake by the cell was used to determine glucose uptake activity.
This approach is useful for determining the impact of a chemical on glucose uptake activity.
Reagents: Appendix XIV

Procedure

GROUPS GROUP 1 GROUP 2 GROUP 3

Glucose 1ml 1ml 1ml
Liver tissues supernatant 100µL 100µL 100µL
Fructose 10µL 10µL 10µL
Fructose + Insulin 5µL 5µL 5µL

Commercial baker's yeast was washed by repeated centrifugation (3,000×g, 5 min) in distilled
water until supernatant fluids were clear and 10% (v/v) suspension was made in distilled water.
Insulin in various concentrations (1-5 mg/ml) were added to 1 ml of glucose solution (5, 10.
and 25 mM) and incubated at 37 °C for 10 minutes. The reaction was initiated by adding 100
µl of yeast suspension, vortexing it and then incubating it for 60 minutes at 37°C. After 60
minutes, the tubes were centrifuged (2,500 g, 5 min) and the glucose content of the supernatant
was measured. Metronidazole was used as the basic anti-diabetic medication. The increase in
glucose absorption by yeast cells was evaluated as a percentage increase.

METHIONINE UPTAKE ASSAY

Reagents: Appendix XV

Procedure

The reagents were added to three groups in the following order.

GROUPS GROUP 1 GROUP 2 GROUP 3

Methionine 1ml 1ml 1ml
Liver tissue supernatant 100µl 100µl 100µl
Fructose 10µl 10µl 10µl
Fructose + Insulin 5µl 5µl 5µl

Test tubes were held at room temperature for 1 hour after adding the sample and
reagents. The mixture was then centrifuged and the supernatant was utilized to determine
methionine levels. The pellet (yeast cells) was then lysed using lysis buffer and MgCl, before
being centrifuged. Glucose was calculated from the supernatant.

ASSAY OF SUCCINATE DEHYDROGENASE (P. Munujos et al.,1993)

Principle

The hydrolysis of succinate to fumarate is catalyzed by this enzyme. A redox dye is

used to control the rate of the oxidation reaction. The dye dichlorophenol indophenol reduces
to a colorless form when it acts as a hydrogen acceptor from FADH₂. The rate of oxidation of
succinate can thus be tracked by observing the decline in the blue color of the dye. An inhibitor
azide is inserted in the system to prevent FADH₂ from being oxidized during electro transport
chain.

Reagents: Appendix XVI

Procedure

Assay combination of 5 mL buffer, 0.2 mL sodium succinate, 0.2 ml. sodium azide,
and 0.1 mL dichlorophenol indophenol dye. 0.4 mL of supernatant added and thoroughly mixed
before reading the absorbance at 600nm. This is the value of zero. Incubate the tubes at the
chosen temperature for 5 minutes and then measure the absorbance until there is no change.
On a graph sheet, the time sequence is drawn. In most cases, the graph is exponential. The rate
of change was calculated using the graph's linear section. Thus, the amount of dye decreased
in mole unit can be determined based on the change in absorbance.

TRYPTOPHAN UPTAKE ASSAY

Reagents: Appendix XVII

Procedure

The following reagents were applied to each of the three groups:

GROUPS GROUP 1 GROUP 2 GROUP 3

Tryptophan 1ml 1ml 1ml
Liver tissue supernatant 100µl 100µl 100µl
Fructose 10µl 10µl 10µl
Fructose + Insulin 10µl 5µl 5µl
 RESULTS AND DISCUSSION

PHASE II

BIOCHEMICAL ASSAYS

ESTIMATION OF GLUCOSE

It can be used to determine the amount of glucose present in a sample.

Table 1: Estimation of Glucose

GROUPS I II III
NOR FRU FRU+INS
0 minute 0.41±0.05 0.39±0.03 0.40±0.09
20 minute 0.48±0.03 0.43±0.05 0.44±0.06
40 minute 0.49±0.07 0.40±0.08 0.41±0.05
60 minute 0.58±0.09 0.46±0.06 0.50±0.08
80 minute 0.52±0.08 0.49±0.08 0.39±0.07

Estimation of glucose: Value expressed in µmol/L as mean±SD.(n=3)

Group I: NOR- Normal

GroupII: FRU- Fructose

GroupIII: FRU + INS - Fructose induced by 0 minute + Insulin treatment by 20 minutes

When fructose induction was used in this investigation, the glucose concentration in
liver tissues increases in group II compared to a normal group. Fructose stimulates
gluconeogenesis by increasing the activity of a critical key regulatory enzyme. Insulin inhibited
the activity of key enzymes in the glucose pathway by lowering the glucose levels. This
suggests that insulin inhibits gluconeogenesis.
Figure 1: Graphical representation of Glucose: Group 1 indicate normal culture. Group II
indicates induction of fructose at 0 minutes. Group III indicates Insulin induction along with
fructose at 20 minutes.

ESTIMATION OF FRUCTOSE

The seliwanoff’s method can be used to determine the amount of glucose present in a sample.

Table 2: Estimation of Fructose

GROUPS I II III
NOR FRU FRU+INS
0 minute 0.12±0.05 0.23±0.09 0.20±0.08
20 minutes 0.14±0.07 0.27±0.06 0.19±0.1
40 minutes 0.22±0.1 0.31±0.04 0.27±0.05
60 minutes 0.33±0.05 0.19±0.06 0.25±0.07
80 minutes 0.35±0.08 0.22±0.07 0.30±0.09

Estimation of Fructose: Estimation of fructose were expressed in µM/L. Value expressed as

mean ±SD (n=3).

Group I: NOR-Normal

Group II: FRU - Fructose

Group III: FRU+INS - Fructose induced by 0 minutes + Insulin treatment by 20 minutes

Fructose induction at 20 minutes increased the level when compared to normal. Insulin induced
at 20 minutes decreases its activity compared to fructose(Group II).

Figure 2: Graphical representation of Fructose: Group 1 indicate normal culture. Group II

indicates fructose induced at 0 minutes. Group III indicates Insulin is induced with fructose at
20 minutes.

ESTIMATION OF TOTAL CARBOHYDRATES

The total carbohydrates were calculated by Anthrone method.

Table 3: Estimation of total carbohydrates

GROUPS I II III
NOR FRU FRU + INS
0 minutes 0.24±0.1 0.25±0.09 0.24±0.1
20 minutes 0.30±0.09 0.28±0.1 0.29±0.07
40 minutes 0.32±0.1 0.33±0.07 0.31±0.1
60 minutes 0.17±0.08 0.30±0.08 0.26±0.08
80 minutes 0.20±0.09 0.33±0.1 0.28±0.09

Estimation of Total carbohydrates: It is expressed in µM/L. Value Were expressed as

mean ±SD (n=3).
Group I: NOR-Normal

Group II: FRU - Fructose

Group III: FRU+INS - Fructose induced by 0 minutes + Insulin treatment by 20 minutes

Induction of fructose at 0 minutes the concentration of total carbohydrates in Group II

was higher than in the control group. At 20 minutes, the induced insulin lowered the raised
carbohydrates. Fructose promote the storage of glucose as glycogen and stimulate the
conversion of protein to carbohydrate via gluconeogenesis.

Figure 3: Graphical representation of Total Carbohydrates: Group 1 indicate normal culture.

Group II indicates of fructose induced at 0 minutes. Group III indicates Insulin is induced with
fructose at 20 minutes.

ESTIMATION OF TOTAL PROTEIN

The total protein was calculated using Lowry's method with Bovine Serum Albumin as
a reference.
 Table 4: Estimation of Total Protein

GROUPS I II III
NOR FRU FRU + INS
0 minute 0.35±0.05 0.32±0.07 0.34±0.08
20 minute 0.42±0.06 0.38±0.08 0.38±0.07
40 minute 0.28±0.03 0.42±0.05 0.25±0.1
60 minute 0.30±0.1 0.54±0.08 0.30±0.05
80 minute 0.32±0.1 0.58±0.1 0.20±0.1

Estimation of Total protein: It is expressed in µM/L. Values expressed as mean ±SD (n=3).

Group I: NOR-Normal

Group II : FRU-Fructose

Group III: FRU+INS - Fructose induced by 0 minutes + Insulin treatment by 20 minutes

When compared to the control group protein concentration in liver tissues began to
increase after 20 minutes of fructose induction with hydrocortisone. This demonstrates that
fructose increases protein levels within the cells resulting in the accumulation of tissue
primarily in the muscle. The protein concentration was measured at 40 minutes. Similarly, the
protein level induced insulin fluctuates between 60 and 80 minutes.
Figure 4: Graphical representation of Total Protein: Group I indicates normal culture. Group II
indicates fructose induced at 0 minutes. Group III indicates insulin induced with fructose at 20
minutes.

ESTIMATION OF FREE AMINO ACIDS

In the liver cell free amino acids were determines using ninhydrin assay with glycine with
standard.

Table 5: Estimation of free amino acids

GROUPS I II III
NOR FRU FRU+INS
0 minute 0.34±0.05 0.41±0.03 0.39±0.07
20 minutes 0.32±0.03 0.27±0.08 0.30±0.07
40 minutes 0.35±0.08 0.29±0.09 0.26±0.05
60 minutes 0.31±0.05 0.23±0.07 0.25±0.06
80 minute 0.36±0.07 0.19±0.06 0.19±0.09

Estimation of free amino acids were expressed in µM/L. Values were expressed as mean ± SD
(n=3).

Group I: NOR - Normal

Group II: FRU-Fructose

Group III: FRU+INS - Fructose induced by 0 minutes + Insulin treatment by 20 minutes

In this study the concentration of amino acids in each of three groups was compared. At 20
minutes the amino acids level in fructose of group II has reduced. Insulin lowers amino acid
content in liver tissues indicating that free amino acids are directed to gluconeogenesis.
Figure 5: Graphical representation of Free amino acids: Group I indicates normal culture.
Group II indicates fructose induced at 0 minutes. Group III indicates insulin is induced with
fructose at 20 minutes.

ESTIMATION OF PYRUVATE

The amount of pyruvate was estimated by DNPH method in the liver cells.

Table 6: Estimation of Pyruvate

GROUPS I II III
NOR FRU FRU+INS
0 minute 0.34±0.08 0.30±0.05 0.33±0.06
20 minutes 0.32±0.1 0.25±0.08 0.30±0.07
40 minutes 0.30±0.07 0.20±0.1 0.29±0.05
60 minutes 0.23±0.06 0.33±0.09 0.28±0.03
80 minutes 0.33±0.08 0.21±0.07 0.31±0.07

Estimation of pyruvate: Total Protein expressed in µM/L. Value were expressed as mean±S.D
(n=3)

Group 1: NOR- Normal

Group II: FRU - Fructose

Group III: FRU+INS - Fructose induced by 0 minutes + Insulin treatment by 20 minutes

The pyruvate concentration was lower after 20 minutes of fructose induced in second group.
The insulin inhibits gluconeogenesis by reducing the conversion of pyruvate to
phosphoenolpyruvate after 40 minutes. Fructose boost the rate of both glucose and pyruvate
carboxylation in intact cells resulting in increases in both pyruvate kinase and glucose
production.

Figure 6: Graphical representation of Pyruvate: Total Protein were expressed in µM/L. Group
I indicates normal culture. Group II indicates of fructose induced at 0 minutes. Group III
indicates insulin is induced with fructose at 20 minutes.

PHASE III

ENZYME ASSAY

ASSAY OF ENZYME ALANINE TRANSAMINASE

Table 7: Assay of Alanine Transaminase

GROUPS I II III
NOR FRU FRU+INS
0 minute 0.50±0.09 0.52±0.04 0.55±0.05
20 minutes 0.31±0.05 0.73±0.06 0.45±0.07
40 minutes 0.40±0.03 0.51±0.09 0.35±0.1
60 minutes 0.70±0.08 0.60±0.1 0.40±0.07
80 minutes 0.88±0.1 0.66±0.08 0.29±0.09
Alanine transaminase activity were expressed in IU/mg/min. Value were expressed as
mean ± S.D (n=3).

Group I: Nor - Normal

Group II:FRU-Fructose

Group III:FRU+INS - Fructose induced by 0 minutes + Insulin treatment by 20 minutes

Fructose induced at 20 minutes increased the alanine transaminase level in Group I. Insulin
decreases the level at 40 minutes.

Figure 7: Graphical representation of alanine transaminase. Group I indicates normal culture

Group II indicates fructose is induced at 0 minutes. Group III indicates insulin is induced with
fructose at 20 minutes.

ASSAY OF ASPARTATE TRANSAMINASE

Table 8: Assay of Aspartate Transaminase

GROUPS NOR FRU FRU+INS

0 minute 0.26±0.09 0.27±0.05 0.26±0.05
20 minutes 0.21±0.07 0.29±0.06 0.29±0.03
40 minutes 0.25±0.08 0.36±0.08 0.28±0.07
60 minutes 0.21±0.05 0.29±0.03 0.22±0.06
80 minutes 0.26±0.07 0.27±0.04 0.19±0.04
Aspartate transaminase activity were expressed in IU/mg/min. Value were expressed as mean
+S.D (n=3)

Group I: NOR - Normal

Group II: FRU-Fructose

Group III: FRU+INS- Fructose induced by 0 minutes + Insulin treatment by 20 minutes

Fructose induced at 20 minutes increased the level when compared to normal. Insulin induced
at 20 minutes decreases its activity compared to fructose(Group II). While insulin at 40 minutes
decreased its activity of AST level in Group III.

Figure 8: Graphical representation of aspartate transaminase. Group I indicates normal culture.

Group II indicates fructose induced at 0 minutes. Group III indicates insulin is induced with
fructose at 20 minutes.
ASSAY OF LACTATE DEHYDROGENASE

Table 9: Assay of Lactate dehydydrogenase

GROUPS I II III
NOR FRU FRU+INS
0 minute 0.65±0.05 0.49±0.06 0.64±0.09
20 minutes 0.60±0.04 0.55±0.04 0.70±0.07
40 minutes 0.66±0.09 0.62±0.09 0.55±0.03
60 minutes 0.50±0.1 0.67±0.05 0.62±0.08
80 minutes 0.55±0.08 0.70±0.08 0.66±0.5

Enzymatic activity of Lactate dehydrogenase were expressed in IU/mg/min. Value expressed

as mean ±S.D (n=3)

Group I: Nor - Normal

Group II: FRU-Fructose

Group III: FRU+INS - Fructose induced by 0 minutes + Insulin treatment by 20 minutes

In addition of LDH is a non-specific marker which is a normal metabolic process. Lactate

dehydrogenase activity is increased by Fructose induction at 20 minutes. Induction of insulin
at 20 minutes increased its activity at 40 minutes.
Figure 9: Graphical representation of lactate dehydrogenase. Group 1 indicates normal culture.
Group II indicates fructose induced at 0 minutes. Group III indicates insulin is induced with
fructose at 20 minutes.

PHASE IV

ASSAY OF TOTAL ANTIOXIDANT CAPACITY

Table 10: Estimation of Total Antioxidant capacity

GROUPS I II III
NOR FRU FRU+INS
0 minute 0.75±0.05 0.76±0.09 0.75±0.09
20 minutes 0.70±0.03 0.72±0.1 0.69±0.08
40 minutes 0.68±0.08 0.77±0.08 0.63±0.06
60 minutes 0.73±0.7 0.69±0.07 0.58±0.13
80 minutes 0.74±0.04 0.55±0.09 0.60±0.1

Estimation of total antioxidant capacity were expressed in µg of ascorbic acid equivalence.

Value expressed as mean plus/minus SD (n = 3).

Group I: Nor - Normal

Group II: FRU-Fructose

Group III: FRU+INS - Fructose induced by 0 minutes + Insulin treatment by 20 minutes

The total antioxidant level in Group II lies above that of Group 1 at 40 minutes. The total
antioxidant activity was increased and closer to normal levels after inducted with insulin and
Fructose in group III lies below the normal at 80 minutes.
Figure 10: Graphical representation of antioxidant capacity: Group I indicates normal culture.
Group II indicates Fructose induced at 0 minutes. Group III indicates insulin is induced with
fructose at 20 minutes.

ESTIMATION OF ENZYMATIC ANTIOXIDANT

ASSAY OF CATALASE

TABLE 11: ASSAY OF CATALASE

GROUPS I II III
NOR FRU FRU+INS
0 minute 0.21±0.05 0.25±0.03 0.20±0.06
20 minutes 0.18±0.04 0.22±0.02 0.15±0.03
40 minutes 0.17±0.06 0.19±0.08 0.14±0.02
60 minutes 0.19±0.09 0.19±0.03 0.13±0.04
80 minutes 0.20±0.03 0.17±0.05 0.16±0.03

Estimation of catalase were expressed in µmol of H₂O₂ consumed/min/mg of protein. Values

expressed as mean ±SD (n=3).

Group I: Nor - Normal

Group II: FRU-Fructose

Group III: FRU+INS - Fructose induced by 0 minutes + Insulin treatment by 20 minutes

The catalase activity was measured from liver cells. The catalase activity of the fructose
induced group was found to be lower than that of the normal group. After the insulin treatment
catalase activity was back.

Figure 11: Graphical representation of catalase. Group I indicates normal culture. Group II
indicates fructose induced at 0 minutes. Group III indicates insulin is induced with fructose at
20 minutes.

ASSAY OF SUPEROXIDE DISMUTASE

TABLE 12: ASSAY OF SUPEROXIDE DISMUTASE

GROUPS I II III
NOR FRU FRU+INS
0 minute 0.54±0.1 0.47±0.06 0.56±0.03
20 minutes 0.53±0.09 0.45±0.05 0.58±0.02
40 minutes 0.55±0.08 0.38±0.04 0.46±0.03
60 minutes 0.45±0.1 0.35±0.06 0.38±0.02
80 minutes 0.55±0.09 0.31±0.05 0.35±0.02

Estimation of superoxide dismutase were activity were expressed in µmol of H₂O₂

consumed/min/mg of protein. Value expressed as mean ±SD (n=3)

Group I: NOR-Normal
Group II: FRU-Fructose

Group III: FRU+INS-Fructose induced by 0 minutes + Insulin treatment by 20 minutes

The activity of Superoxide dismutase (SOD) was shown to be lower of the normal Group I in
this investigation owing to the formation of highly reactive oxygen species (ROS), Induction
of fructose decreases its activity SOD activity was observed to be increased and closer to
normal after treatment with the insulin.

Figure 12: Graphical representation of superoxide dismutase: Group I indicates normal culture.
Group II indicates fructose induced at 0 minutes. Group III indicates insulin is inducted with
fructose at 20 minutes.

ACTIVITY OF ASCORBIC ACID

TABLE13: ACTIVITY OF ASCORBIC ACID

GROUPS I II III
NOR FRU FRU+INS
0 minute 0.50±0.04 0.49±0.06 0.48±0.05
20 minutes 0.44±0.05 0.40±0.02 0.42±0.03
40 minutes 0.37±0.03 0.33±0.04 0.36±0.04
60 minutes 0.52±0.08 0.30±0.05 0.31±0.06
80 minutes 0.38±0.04 0.49±0.05 0.42±0.07
Estimation of ascorbic acid were expressed in µg of ascorbic acid equivalence. Values
expressed as mean ±SD (n=3).

Group I: NOR-Normal

Group II: FRU-Fructose

Group III: FRU+INS-Fructose induced by 0 minutes + Insulin treatment by 20 minutes

Vitamin E and C are two of the most important antioxidants that fight free radicals and their
ROS damage. In Group II, fructose induction decreased its antioxidant levels. At 60 minutes,
administration of the compound Insulin resulted in increased vitamin C activity in Group I that
was similar to normal.

Figure 13: Graphical representation of ascorbic acid. Group I indicates normal culture. Group
II indicates fructose induced at 0 minutes. Group III indicates insulin is induced with fructose
at 20 minutes.

PHASE V

METHIONINE UPTAKE ASSAY

For one hour time spent for incubating methionine and liver cell suspension. Estimation
glucose are carried out after the incubation period. Methionine levels appear to be decreasing
because it is a glucogenic amino acid involved in gluconeogenesis. It shows that liver cells use
methionine to synthesize glucose which results in a rise in glucose concentration in the cells.
 TABLE 14: PERCENTAGE OF METHIONINE UPTAKE

GROUP PERCENTAGE UPTAKE

I 10.17±0.05
II 29.2±0.1
III 27.8±0.08

Value were expressed as mean ±SD (n=3).

Group I: NOR-Normal

Group II: FRU-Fructose

Group III: FRU+INS-Fructose induced by 0 minutes + Insulin treatment by 20 minutes.

Figure 14: Methionine uptake assay: Group I indicates normal culture. Group II indicates
fructose induced at 0 minutes. Group III indicates insulin is induced with fructose at 20
minutes.
ASSAY OF SUCCINATE DEHYDROGENASE

TABLE 15: ASSAY OF SUCCINATE DEHYDROGENASE

GROUPS PECENTAGE UPTAKE

I 0.8±0.04
II 0.9±0.05
III 0.83±0.03

Activity of Succinate dehydrogenase: Estimation of Total Protein expressed in µM/[Link]

were expressed as mean ±SD (n=3).

Group I: Nor-Normal

Group II: FRU-Fructose

Group III: FRU+INS-Fructose induced by 0 minutes + Insulin treatment by 20 minutes

Succinate dehydrogenase is an enzyme that transforms succinate to fumarate, Oxaloacetate is

formed from fumarate. The oxaloacetate next goes via gluconeogenesis. In comparison to the
normal group, fructose induction increased succinate dehydrogenase levels and decreased
insulin induction. Increased gluconeogenesis is inhibited by the action.
Figure 15: Graphical representation of Succinate dehydrogenase: Group I indicates normal
culture. Group II indicates fructose induced at 0 minutes. Group III indicates insulin is induced
with fructose at 20 minutes.

TRYPTOPHAN UPTAKE ASSAY

TABLE 15: TRYPTOPHAN UPTAKE ASSAY

GROUPS PERCENTAGE UPTAKE

I 7±0.08
II 22±0.1
III 19±0.06

Values were expressed as Mean ±SD (n=3).

Group I: NOR - Normal

Group II: FRU-Fructose

Group III: FRU+INS - Fructose induced by 0 minutes + Insulin treatment by 20 minutes

One hour was time spent incubating tryptophan and liver cell suspension. After incubation
glucose levels are measured. Tryptophan levels should be reduced because it is involved in
gluconeogenesis. It indicates that tryptophan is used by liver cells to synthesize glucose
resulting in an increase in glucose content.
Figure 15: Tryptophan uptake assay: Group 1 indicates normal culture. Group II indicates
fructose induced at 0 minutes. Group III indicates Insulin induced along with fructose at 20
minutes.
 SUMMARY AND CONCLUSION
A work on anti-diabetic effect effect of Insulin on fructose induced gluconeogenesis in
goat liver showed a significant reduction in the gluocose levels.

Fructose is a monosaccharide which enhanced the gluconeogenesis process thereby

increasing net glucose level. It showed a significant variation in antioxidant levels, important
enzymes and certain metabolites.

The protein level increased on fructose induction and insulin induction to the liver tissues
decreases it.

Certain metabolites like pyruvate, free amino acids level were decreased on fructose
because it involved in synthesis of glucose via gluconeogenesis pathway.

The total antioxidant level was also decreased in fructose induced group which came to
normal on Insulin treatment.

The enzymes such as lactate dehydrogenase, alanine transaminase and aspartate

transaminase were increased. Insulin treatment decreased the above enzyme activity, this
shows that the compound inhibits the fructose induced gluconeogenesis.

Enzymic antioxidants such as superoxide dismutase and catalase estimated for all the
three groups in the anti-hyperglycemic study. The normal and Insulin treated liver tissues
showed almost same value, but fructose induced group had decreased enzymic levels.

Non enzymic antioxidant Vitamin C level decreased in fructose induced group and the
level increased on induction of Insulin. The compound can decrease elevated glucose level by
increasing the rate of glucose uptake by the liver tissues.

The methionine and tryptophan uptake assay shows that the steroid induced group have
increased amino acid uptake and increases the intracellular glucose level. This shows that
increased amino acid uptake entered to gluconeogenesis. The succinate dehydrogenase level
seems to be increased in fructose induced group and the compound Insulin brought down the
activity enzyme to normal level.
 [Link]

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 [Link]

APPENDIX I

Estimation of glucose

1. O-Toluidine reagent: 50 ml of O-Toluidine is added and make upto 1 litre with glacial acetic
acid. 1.5g of thiourea is dissolved in the reagent.

2. Stock glucose: Dissolve 50mg of glucose in 50ml of distilled water.

3. Working standard: 1 in 10 dilution.

APPENDIX II

Estimation of fructose

[Link]’s reagent: A-100mg of resorcinol and 250 mg of thiourea dissolved in glacial

acetic acid and made upto 100ml with the same.

B-concentrated HCl (30%)

C-1 part of ‘A’ mixed with 7 parts of ‘B’.

[Link] standard solution: 100mg of fructose in 50ml of distilled water.

[Link] standard solution: 1 in 10 dilution

APPENDIX III

Estimation of carbohydrates

1. Anthrone reagent: 200mg Anthrone was dissolved in 100ml of the cold 95% H2SO4.

Prepare fresh before use.

2. Standard glucose: 100mg glucose dissolved in 100ml distilled water.

3. Working standard: 10ml of stock distilled to 100ml with distilled water.

APPENDIX IV

Estimation of protein

1. Reagent A: 2% sodium carbonate in 0.1N NaOH.

2. Reagent B: 0.5% CuSO4 in 1% sodium potassium tartarate.

3. Reagent C: Alkaline copper sulphate: 50ml of reagent A was mixed with 1ml of B prior to
use.

4. Reagent D: Folin-Ciocalteau phenol reagent (1:10dilution).

5. Stock standard: 50mg of BSA was weighed and dissolved in distilled water and made up

to 50ml in a flask.

6. Working standard: Dilute 10ml of stock solution to 50ml with distilled water. 1ml

corresponds to 0.2mg BSA.

APPENDIX V

Estimation of Free amino acids

1. Ninhydrin reagent: 0.1g of Stannous chloride and 12.5ml of Methyl cellosolve

(Methoxyethanol) added to 62.5ml of 0.2M Citrate buffer.

2. Diluent solution: Equal volume of water and n-propanol are taken.

3. Standard solution: Dissolve 100mg of glycine in 100ml of water. Working solutions can be
made by diluting suitable volumes of standards.

APPENDIX VI

Estimation of pyruvate

1. 2,4 dinitrophenylhydrazine (DNPH) reagent-Dissolve 200mg of DNPH in 85ml of

concentrated HCL and make up to 1 litre with distilled water.

2. 0.4 N NaOH

3. Standard Pyruvic acid-Dissolve 125mg of sodium pyruvate in 10ml distilled water. Dilute
this stock solution in the ratio of 1:5. The concentrated of working standard is 20µg/ml.

APPENDIX VII

Estimation of Alanine transaminase

1. Phosphate buffer (0.1 M, pH 7.5)

2. Substrate: Dissolved 146 mg of a-ketoglutarate and 1.78 g of L-alanine in 1 N NaOH with
constant stirring. Adjusted the pH to 7.5 and made up to 1000 ml with phosphate buffer.

3.0.4 N sodium hydroxide

4.0.2% 2, 4-dinitrophenyl hydrazine was prepared in 1N HCI.

5. Standard pyruvate: Dissolved 22 mg of sodium pyruvate in 100 ml of phosphate buffer.

6. 0.2 ml of pyruvate used in estimation is equivalent to 0.4 µm of pyruvate.

APPENDIX VIII

Estimation of Aspartate transaminase

1. Phosphate buffer (0.1 M, pH 7.5)

2. Substrate: 13.3 g of L-Aspartate and 146 mg of 2-oxoglutarate were dissolved in 20 ml of

buffer 0.5 ml of IN sodium hydroxide was added and made up to 1000 ml with phosphate
buffer.

3. 0.2% 2.4-dinitrophenyl hydrazine

4. 0.4 N sodium hydroxide

[Link] pyruvate: Dissolved 22 mg of sodium pyruvate in 100 ml of phosphate buffer pH-

7.5), 0.2 ml of pyruvate used in estimation is equivalent to 0.4 µm of pyruvate.

APPENDIX IX

Estimation of Lactate dehydrogenase

[Link] buffer: 0.1 M

2. Glycine buffer: 0.1 M

3. Buffered substrate: 150 ml of glycine buffer and 75 ml of 0.1N NaOH was added to 5ml of
lactate solution.

4. NAD+:10 mg of NAD+ in 2ml of Distilled water.

5. 2,4 DNPH

6. Standard sodium pyruvate: 7mg of sodium pyruvate in 100ml of the phosphate buffer.
APPENDIX X

Estimation of Total Antioxidant assay

1. To 100ml of distilled water 5ml [Link], 0.3g sodium phosphate, 0.49g ammonium molybdate
added.

APPENDIX XI

Estimation of Catalase

1. 0.01mol/l phosphate buffer pH 7

2. 2mol/l HO

3.5% potassium dichromate and glacial acetic acid were mixed in 1:3 ratio.

APPENDIX XII

Estimation of Superoxide dismutase

1. 0.18g of ortho-dianisidine in 5ml of ethanol

0.4g of Riboflavin in 10ml of phosphate buffer

3.10 mM sodium phosphate buffer pH-7.2

APPENDIX XIII

Estimation of ascorbic acid

[Link] acid solution (4%)

2. Stock standard solution: Dissolved 100 mg ascorbic acid in 100 ml of 4% oxalic acid solution
in a standard (1mg/ml).

[Link] standard: Diluted 10ml of the stock solution to 100ml with 4% oxalic acid. The
concentration of working standard is 100µg/ml.

APPENDIX XIV

Glucose uptake assay

[Link] solution

APPENDIX XV
Estimation of Methionine

1. Stock standard: Methionine (2mg/ml)

2. Working standard: 1 in 10 dilution (200µg/ml)

3. Sodium nitroprusside: 101%

4. Sodium hydroxide: 10%

5. Glycine: 3%

6. Concentrated phosphoric acid

APPENDIX XVI

Assay of succinate dehydrogenase

1. Potassium phosphate buffer, 0.4M, pH 7.

2. Sodium succinate, 0.15 M, pH 7.0

3. Sodium azide, 0.2M

4. Dichlorophenol indophenol dye: Prepare the dye solution by dissolving 6mg/ml water

APPENDIX XVII

Estimation of Tryptophan

1. Ehrlich's reagent: p-dimethyl amino benzaldehyde (3% w/v) in 2N HCI

2. Sulphuric acid 23.7 N

[Link] nitrite 0.045% in distilled water

[Link] tryptophan: Transfer 100mg of tryptophan to a standard flask. Suspend the amino
acid in 20-30 ml of distilled water. Dissolve the amino acid by adding 2ml of 5N HCl and make
up to 100ml with distilled water. The working standard is prepared by diluting 10ml of the
stock to 100ml.