Aegle marmelos (L.) Corr. impedes onset of Insulin ... · Draft 1 Aegle marmelos (L.) Corr. impedes onset of Insulin resistance syndrome in rats provided with drinking fructose from

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Aegle marmelos (L.) Corr. impedes onset of Insulin

resistance syndrome in rats provided with drinking fructose from weaning to adulthood stages of development: A

mechanistic study

Journal: Canadian Journal of Physiology and Pharmacology

Manuscript ID cjpp-2016-0236.R1

Manuscript Type: Article

Date Submitted by the Author: 12-Sep-2016

Complete List of Authors: MATHUR, RAJANI; Delhi Institute of Pharmaceutical Sciences and Research, Pharmacology; Delhi Institute of Pharmaceutical Sciences and Research SEHGAL, RATIKA; Delhi Institute of Pharmaceutical Sciences and Research, PHARMACOLOGY RAJORA, PREETI; Delhi Institute of Pharmaceutical Sciences and Research SHARMA, SHVETA; Delhi Institute of Pharmaceutical Sciences and Research KUMAR, RAJESH ; Delhi Institute of Pharmaceutical Sciences and Research

MATHUR, SANDEEP; All India Institute of Medical Sciences, PATHOLOGY

Keyword: Aegle marmelos, fructose, GLUT 2, JAK-STAT3, Insulin resistance syndrome

https://mc06.manuscriptcentral.com/cjpp-pubs

Canadian Journal of Physiology and Pharmacology

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1

Aegle marmelos (L.) Corr. impedes onset of Insulin resistance syndrome in rats provided with

drinking fructose from weaning to adulthood stages of development: A mechanistic study

Rajani Mathur*, Ratika Sehgal, Preeti Rajora, Shveta Sharma, Rajesh Kumar, Sandeep

Mathura

Department of Pharmacology, Delhi Institute of Pharmaceutical Sciences and Research

(DIPSAR), Pushp Vihar, Sec III, M.B. Road, New Delhi-110017. INDIA

a Department of Pathology, All India Institute of Medical Sciences, Ansari Nagar, New Delhi-

110029, INDIA

*Corresponding Author:

Dr. Rajani Mathur

Assistant Professor

Department of Pharmacology,

Delhi Institute of Pharmaceutical Sciences and Research (DIPSAR),

Pushp Vihar, Sec III, M.B. Road, New Delhi-110017. INDIA

Phone: 011-29554327

Fax: 011-29554503

e-mail: [email protected]

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ABSTRACT

To explore the effect of aqueous extract of leaves of Aegle marmelos (AM) on hepatic

carbohydrate metabolism and insulin downstream signalling in rats provided with drinking

fructose (15%) from weaning to adulthood. Wistar albino rats (4week) were randomly

divided into Normal Control (NC), Fructose Control (FC) and treatment (AMT) groups and

provided over 8 weeks, chow + water, chow + fructose (15%) and chow + fructose (15%) +

AM (500 mg/kg/d, p.o.), respectively. Significantly (p<0.05) raised levels of Fasting Blood

Glucose, lipid, visceral weight, plasma insulin and leptin, glycogen, gluconeogenesis enzyme

levels but decreased glycolytic enzyme activity was recorded in FC as compared to NC.

Raised levels of glucose transporter (GLUT 2) protein but decreased activity of

phosphatidylinositol-3-kinase (PI3K/AkT) and Janus Kinase –Signal Transducer And

Activator of Transcription-3 (JAK-STAT3) in hepatic tissue, indicate a state of insulin and

leptin resistance in FC. AMT recorded significant (p<0.05) lowering of physical, and

glycemic parameters, reinforcement of hepatic glycolytic over gluconeogenic pathway and

upregulated PI3K/AkT and JAK-STAT3 pathways, as compared to FC. For the first time, the

mechanism underlying development of Insulin Resistance Syndrome (IRS) is delineated here,

along with the potential of Aegle marmelos in impeding the same.

KEY WORDS

Aegle marmelos; fructose; gluconeogenesis; Glucose Transporter 2 (GLUT 2); JAK-STAT3;

PI3K/AKT; Rutin; Insulin resistance syndrome

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INTRODUCTION

Insulin resistance syndrome (IRS) is a multi-component pre-diabetic state where

hyperlipidemia and hyperglycemia is associated with increased risk of type-II diabetes,

hypertension, neurodegenerative disorders and cardiovascular diseases (Alberti et al. 2005).

About 25% of the world’s adult population in both developed and developing countries

suffers from IRS (IDF 2005) and the tally is projected to rise up to 40% by 2025 (Rinaudo et

al. 2012). It is now evident from experimental, cross sectional and prospective studies that a

range of molecular, cellular, metabolic, neuroendocrine and physiological adaptations occur

in response to nutritional environment during pre-pubertal and pubertal years that may

manifest as IRS in adult life (Cruz et al. 2007; Mcmillen et al. 2005; Srinivasan et al. 2002).

Fructose is a simple carbohydrate with a low glycemic index. When consumed for

medium to long-term duration, it contributes to tissue insulin insensitivity, pronounced

metabolic defects often without obesity, and pathogenesis of IRS, a pre-diabetic state

(Basicano et al. 2005;Miller et al. 2002). Fructose is extensively used as high fructose corn

syrup (HFCS) in carbonated beverages, baked goods, canned fruits, and some dairy products.

It is reported that children derive as much as 10% of their daily energy via consumption of

fructose (~ 30-40 g/day) (Park et al. 1993). A longitudinal study on children (6-13 years)

showed that drinking excessive amounts of sweetened drinks (> 12 oz/day) correlated with

high daily energy intake, that initiated a cascade which culminated as IRS by the time they

reach adulthood (Totty 2010).

The mechanistic pathogenesis of IRS is poorly delineated. Further, there exists severe

paucity of preventive/therapeutic modalities that can address the underlying mechanisms. It is

grossly elucidated that following hepatic absorption via glucose transporter 2 (GLUT 2)

(Sharawy et al. 2016), excess fructose is metabolized unhindered by fructokinase to fructose-

1-phosphate and finally glycogen via pathways of gluconeogenesis (Bjorkman et al. 1989;

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Tappy et al. 2010). Thus, a pattern of hyperglycemia accompanied by compensatory

hyperinsulinemia is initiated that ultimately disrupts the insulin-signalling pathway.

Resistance of the intracellular insulin signalling pathway acting through Phosphatidylinositol

3-kinase/Akt (PI3-K/Akt) transducers, is understood to be the central molecular mechanism

leading to IRS (Wang et al. 2016).

Since ancient times, plants have been attributed with medicinal properties and used

for the treatment of variety of diseases. Leaves of Aegle marmelos (L.) Correa (fam:

Rutaceae) have been well documented for its anti-diabetic, anti-hyperglycemic and anti-

hyperlipidemic (Upadhya et al. 2004) properties but not for management of IRS. Although

the leaves have been analysed extensively to show presence of various phytochemicals, such

as skimmianine, aegeline, rutin, lupeol, cineol, citral, citronella, cuminaldehyde, eugenol,

marmesinine, the pharmacologically active phyotchemical has not been identified, till date

(Maity et al. 2009).

The major impediment in deriving the full potential of medicinal plants, is the

challenge in authenticating them by using modern analytical tools to develop their finger

print. It has been unanimously decided by global agencies like World Health Organization

(WHO), United Nations Industrial Development Organization (UNIDO), International

Certification Services (ICS) to use phytochemical reference standard (PRS), which may

either be therapeutically active compound or any compound unique to the plant or its major

phytochemical constituent, for developing assay method to authenticate the herbal drug

(Medicinal Plants Unit, 2010). It has been reported earlier that the content of flavonoids and

phenolic compounds in the leaf extract of A. marmelos is very high (~8.248 ± 0.02 mg/kg)

(Siddique et al. 2010). So, in the present study, rutin, one of the major flavonol known to be

present in leaves of Aegle marmelos, was identified as PRS for developing assay method to

authenticate the extract for pharmacological assessment.

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Thus, the present study explores the protective effect, if any, of aqueous extract of

leaves of Aegle marmelos (500 mg/kg/d) against the metabolic correlates of IRS that manifest

when developing rats are provided fructose (15%), ad libitum, over 8 weeks.

MATERIALS AND METHODS

Plant Collection, Preparation and Phytochemical Analysis

Fresh leaves of Aegle marmelos (Linn.) correa (Family-Rutaceae) were collected

locally between February-March. Leaves were taxonomically identified and authenticated by

Principal Scientist- National Bureau of Plant Genetic Resources (Indian Council of

Agricultural Research) Pusa campus, New Delhi, India and the voucher specimen is

deposited at the herbarium of NISCAIR, New Delhi (NISCAIR/RHMD/consult/-2010-

11/1536/134).

The leaves of Aegle marmelos were washed with fresh water and dried in shade. The

dried leaves were powdered and mixed with distilled water (1:10) and shaken for 2 h on a

mechanical shaker and filtered. The filtrate was frozen at -30°C and lyophilized at -70°C to

obtain aqueous extract of leaves of Aegle marmelos (AM). AM was subjected to standard

qualitative assays for the presence of alkaloids, flavonoids, terpenoids and steroids.

HPLC Conditions for Quantification of Rutin Content in AM

Rutin was quantitatively estimated in AM by reversed phase high performance liquid

chromatography (RP-HPLC) using Prominence UFLC system (Shimadzu Corporation,

Kyoto, Japan) connected by Prominence communication bus module (CPM-20, Shimadzu

Corporation, Kyoto, Japan) to data processing software, LC solutions (Shimadzu

Corporation, Kyoto, Japan) installed in windows 7 operating system. The chromatographic

separation was performed using isocratic elution with mobile phase consisting of

methanol:water (1:1) with pH adjusted to 2.8 with ortho-phosphoric acid, pumped by

Prominence liquid chromatography (LC-20AD, Shimadzu Corporation, Kyoto, Japan) at a

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flow rate of 1.0 ml/min in a reverse phase analytical enable C-18 G column (250 x 4.6 mm

I.D., S/N WL06-082), The system was maintained at room temperature which yielded a

column back pressure of 2200-2400 psi. Prior to injecting samples, the column was

equilibrated for at least 30 min with the mobile phase. The samples were injected by

Hamilton syringe (Microlitre TM #702, 0.0025 ml) into the 20 µl loop Rheodyne manual

sample injector with switch (Rheodyne®

Injector P/N 7725i), ran for 10 min in the column

and the effluent was monitored at 256 nm by Prominence diode-array detector (SPD-M20A,

Shimadzu Corporation, Kyoto, Japan). All chromatographic data was recorded and processed

using LC solution software.

Preparation of Standard Stock and Sample Solution of Rutin and AM

The standard stock solution (1mg/ml) of rutin (LOBA Chemie, Mumbai, India,

>99.99% pure) was prepared in ethanol and ultra-sonicated for 5 min followed by filtration

through 0.45 µm nylon membrane filter and further diluted with the mobile phase to give a

working concentration range of 5-80 µg/ml.

Plotting Calibration Curve

Calibration curve was constructed by analyzing the series of standard rutin

concentration in the range of 5-80 µg/ml. Each concentration was injected in triplicate for

three consecutive days for intra-day and inter-day precision. Rutin content in AM was

determined by external spiking method. The rutin peak in sample was identified using in-built

software that is based on matching retention time and spectra of the peaks. The method was

developed and validated in-house based on parameters such as linearity, limit of detection,

limit of quantification, accuracy and precision.

Experimental Design

In accordance with the approved protocol by the Institutional Animal Ethics

Committee (IAEC/DIPSAR/2013/06), experimental rats were housed in polycarbonate cage

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under standard conditions; temperature (22±2°C), humidity and dark/light cycle (12/12 h).

Experimental animals were cared for, used and sacrificed in accordance with the Guide to the

Care and Use of Experimental Animals as set by the Canadian Council on Animal Care

(www.ccac.ca) and Committee for the Purpose of Care and Supervision of Experiments on

Animals (CPCSEA, www.cpcsea.nic.in).

Weaned male wistar albino rats (4 weeks old), were randomly divided into 3 different

groups (n=6 each), normal control (NC), fructose control (FC) and treatment (AMT). All the

groups received pre-weighed standard laboratory chow diet (Pranav Agro Ltd., India), the

composition (%w/w) of which was- crude protein (20-22), crude fat (4-5), crude fiber (6),

moisture (8-9), calcium (1.2), phosphorous (0.6-0.8), to provide 3600 Kcal of

metabolizableenergy per Kg of pellet diet. The animals in the NC, FC and AMT groups

received either filtered water or fructose(15%) ad libitum, as drinking solutions. Freshly

reconstituted AM (500 mg/kg/d, p.o) was administered to AMT, over study period.

Effect on Food/Water/Fructose Intake

The animals were provided with pre-measured standard laboratory chow diet and

filtered drinking water/fructose (15%) ad libitum and the amount consumed over 24 h was

measured. Using this data, total calorie intake (kcal of metabolizing energy/gm feed intake +

kcal of energy /gm fructose) of each group over the study duration was calculated.

The body weight of each animal was recorded daily, using a sensitive digital balance.

After 8 weeks, the animals were anaesthetized (Ketamine:Xylazine:80 mg/kg:10 mg/kg ip),

and their organs (liver, heart, kidney and intestine) were surgically removed, washed with

normal saline (0.9%), dabbed dry on blotting paper and weighed.

Fasting Blood Glucose and Oral Glucose Tolerance Test

On weekly basis, fasting blood glucose (FBG) of all the animals was recorded after a

period of food deprivation but with free access to filtered drinking water, spanning over 12 h.

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The tail tip of the animals was pricked using a sterile needle and FBG concentration was

recorded using digital glucometer (Accu check active, Roche, USA).

Oral glucose tolerance test (OGTT) was conducted at the start and end of the study

using standard protocol (Du Vigneaud and Karr 1925). After food deprivation for 12 h, the

animals were orally loaded with glucose (2 g/kg) and blood glucose concentration (mg/dl)

was recorded at 0 (before glucose administration), 15, 30, 60, 90 and 120 min after glucose

administration. The plasma glucose concentration was plotted over the time points and

integrated area under the curve (AUC) was computed.

Biochemical Estimations

On completion of the study period the animals were anaesthetized

(Ketamine:Xylazine::80 mg/kg:10 mg/kg i.p.) and the blood was collected by cardiac

puncture. In the separated plasma, fasting insulin and leptin levels in plasma, were estimated

using Elisa based immunoassay kits, in accordance with the instruction manual provided by

the manufacturer (Raybio, USA). Homeostasis Model Assessment (HOMA)-index of IR was

calculated using the formula: HOMA IR = 0.062 × glucose level (mg/dl) × insulin (ng/ml).

Separated serum was subjected to biochemical lipid profiling {triglycerides (TG),

total cholesterol (TC), high density lipoprotein (HDL), low density lipoprotein (LDL), very

low density lipoprotein (VLDL)} using routine protocols.

Hepatic Carbohydrate Metabolism Enzyme Estimations

Liver from all the anaesthetized animals was excised, and partly sectioned and fixed

(10% formalin) for histopathological processing and partly stored at -80°C for biochemical

estimations. Glycogen was estimated as per the anthrone test method (Seifter et al. 1950).

Enzyme activity of hexokinase, fructose -1, 6-bisphosphate and glucose-6-phosphate was

assessed using standard protocols (Koide et al. 1959; Majumder et al. 1982; Murray et al.

1999).

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Hepatic Insulin and Leptin Downstream Signalling Transducer Measurement

Excised liver was perfused with collagenase-calcium free hanks solution till it was

completely blanched. Approximately 100 mg of tissue was weighed and homogenized in 6 ml

of homogenization solution (collagenase calcium free hanks:incubation solution :: 1:1).

Incubation solution constituting of EDTA (20mM), DTT (1mM) in 0.05% v/v β-

mercaptoethanol in Phosphate Buffered Saline (PBS) was prepared. The homogenate was

centrifuged at 500 rpm for 2 min. The supernatant was collected and re-centrifuged at 14,000

rpm for 15 min at 4°C. The obtained pellet was re-suspended in the cell lysis buffer. The

suspension was left undisturbed for 1 h, and then centrifuged (12,000 rpm, 4°C). The

supernatant was collected and assessed for levels of PI3K/Akt and JAK-STAT- phospho as

using Elisa-based-immunoassay (EIA) kits, per the manufacturer’s protocol (RayBio, USA).

Liver Histopathology

Liver sections were fixed in fresh neutral buffered formalin (10%) overnight at room

temperature and then embedded in paraffin. Section (4 µm) were cut using a rotary

microtome and stained in hematoxylin and eosin (H and E) stain for histopathological study.

Steatosis was estimated by percentage of fat in hepatocytes by examination at 40 and 100X.

An experienced pathologist (SM, 25 years of experience), blinded to study results, evaluated

the liver biopsy samples for histopathologic grading of hepatic steatosis. Histologic sections

from liver were arbitrarily graded on percentage of steatosis as mild (1-30%), moderate (30-

70%) and severe (>70%).

Hepatic Immunohistochemistry

Paraffin embedded liver sections were subjected to heat-induced antigen retrieval as

per standard protocol. The sections were rinsed in PBS and blocked in 1% normal goat serum

for 1 h at room temperature. The sections were incubated in rabbit anti-rat GLUT2 (Abcam,

USA) (1:100 dilution) for 16 h at 4°C, washed in PBS, blocked in 1% normal mouse serum

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and incubated for 16 h at 4°C in mouse anti-rat GR. Appropriate secondary antibody was

applied for 1 h at room temperature. The slides were analysed by trained pathologist blinded

to the treatment.

Statistical Analysis

All the results are shown as mean±SEM; (n=6). Student t-test was used to analyze

difference in variables before and after fructose feeding; between fructose control (FC) and

treatment group (AMT); and normal control (NC) and fructose control (FC). p values ≤ 0.05

was considered statistically significant. Statistical analysis was done using GraphPad prism

5.0.1 installed in windows 7 home basic operating system.

RESULTS

Quantification of rutin in AM using RP-HPLC

A sensitive isocratic RP-HPLC method was developed in our laboratory and

standardized with precise interday and intraday values to give well-separated peaks of AM

with LOD and LOQ as 0.18 and 0.55 µg/ml, respectively. Commercially procured rutin

(LOBA Chemie, Mumbai, India, >99.99% pure) was used as external standard to quantify

rutin content in AM. Under these conditions, rutin was detected at Rt=7.12±0.04 min, with

linearity between 5-80 µg/ml (r2=0.998) and calculated to be 8.52 µg/mg of AM (Fig. 1a-b).

Effect of AM on Food/Water/Fructose Intake

Throughout the study, the average weekly food intake was significantly (p<0.001)

lower in FC as compared to NC. The consumption of lab chow diet in AMT was significantly

lower as compared to FC (p<0.05) (Fig 2a). The average weekly fructose intake was lower in

AMT group as compared to FC (Fig 2b). Total calorie intake of animals over 8 weeks study

duration in NC, FC and AMT was 21623.4, 18552.51, 15712.02 kcal/gm respectively.

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At the start of the study, the average body weight of animals in each group, was not

different significantly. During the study, the weight gain pattern in AMT was significantly

lower as compared to FC (p<0.001) and evident upon the completion of 1st week (Fig 2c).

Effect of AM on Fasting Blood Glucose and Oral Glucose Tolerance Test

At eighth week, FBG was significantly (p<0.05) higher in FC (97.40±3.440

mg/dl) than in NC (86.50±3.998 mg/dl). The FBG concentration of AMT (73.17±2.197

mg/dl) was significantly lower (p<0.001) as compared to FC (94.40±3.059 mg/dl) at 5th

week

(Fig 2d).

The AUC’s of OGTT in NC, FC and AMT are 8828.75, 10357.5, and 9006.25

respectively. The mean values of blood glucose concentration were significantly higher in FC

group as compared to NC at time points- 0, 15, 30, 60, 90 and 120 min. A significant

decrease in average blood glucose value of AMT (139.8±2.136 mg/dl) was observed at 30

min (Fig 2e).

Effect of AM on Biochemical Estimations

At the end of 8 weeks, fructose intake lead to significant increase in serum TG and

VLDL levels in FC as compared to NC, that was reduced significantly in AMT. The weight

of the visceral organs (liver, heart, kidney and intestine) was increased in FC as compared to

NC but significantly (p<0.001) reduced in AMT (Table 1).

FC showed a significant (p<0.001) rise in fasting plasma insulin levels after 8 weeks

study period as compared to NC, which was reduced significantly (p<0.001) in AMT. A

significant (p<0.001) increase in HOMA-IR in FC (8.45±2.52) was recorded as compared to

NC (2.41±0.60) but restored in AMT ( 2.53±0.75) (Table 1).

After 8 weeks study period, significant hyperleptinemia was recorded in FC

(595.66±198) as compared to NC (110.6±37.55), but not in AMT ( 42.57±6.06) (Table 1).

Effect of AM on Hepatic Carbohydrate Metabolism

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After 8 weeks study, significant elevation in glycogen levels was recorded in FC

(7.841±0.081 µg/g liver) group as compared to NC (5.729±0.011 µg/g liver). In AMT

(5.508±0.01 µg/g liver) the glycogen content was significantly reduced as compared to FC

(Table 1).

The activity of hexokinase enzyme increased significantly (p<0.001) in the FC

(35.6±0.14 µmoles/10 mg liver) compared to NC (110±2.34 µmoles/10 mg liver). The

enzyme activity was restored around normal values (113.1±3.90 µmoles/10 mg liver) with a

significant increase in activity as compared to FC (Fig 3a).

Glucose-6-phosphatase levels were significantly high in the FC (216.2±2.29 ng/10 mg

liver) as compared to NC (195.8±0.823 ng/10 mg liver). After 8 weeks study period,

enzymatic activity significantly increased in AMT (202.4±1.032 ng/10 mg liver) group (Fig

3b).

Fructose consumption significantly elevated the fructose -1,6- bisphosphatase

enzyme levels in FC (51.5±3.1782 ng/10 mg liver) as compared to NC (12.22±1.2312 ng/10

mg liver) after 8 weeks. A significant (p<0.001) reduction was observed in AMT

(23.51±1.9120 ng/10 mg liver) as compared to FC (Fig 3c).

Effect of AM on Insulin and Leptin Signalling Pathway

A significant (p<0.001) reduction in absorbance (AU) was recorded in FC

(0.2345±0.002941) in JAK STAT 3 expression at the end of the study protocol compared to

NC (0.3103±0.003007) while, AMT (0.2633±0.002076) significantly (p<0.001) increased

these levels (Fig 3d).

The PI3K levels of FC group was significantly (p<0.001) decreased in liver

homogenates as compared to NC. Treatment with AM restored back the values close to NC.

The percentage elevation values for FC (19.28%) decreased by almost 2 fold compared to NC

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(44.93%). AMT group levelled off with the NC group with the percentage elevation of

34.23% (Fig 3e).

The absorbance (AU) of Akt corresponding to its levels in liver decreased in FC

(0.2070±0.001238) as compared to NC (0.2395±0.0009916) after 8 weeks of study period.

The Akt level in AMT was estimated to be 0.1480±0.001713 (Fig 3f).

Effect of AM on Liver Histopathology

Histological sections of liver of NC displayed normal architecture of the

hepatocytes with mild steatosis. Macro and micro vesicular fatty changes were severe in liver

sections of FC. Following treatment with AM, the fatty changes were mild and architecture of

the cells was normal in AMT (Fig 4a-c).

Effect of AM on Hepatic GLUT 2

Immunoreactivity of GLUT 2 as determined by immunohistochemistry on

cellular membranes of NC (Fig 4d) was remarkably lower than FC group (Fig 4e). The

immunoreactivity displayed in AMT (Fig 4f) was sparse and patchy and close to NC group .

DISCUSSION

The lean type metabolic syndrome characterized by marked insulin resistance and

comparatively lesser weight gain is gaining prevalence among children and adolescents

because of preferential intake of fructose-laden beverages and confectionaries (Totty 2010).

At puberty, both non-diabetic and diabetic children, exhibit increased state of insulin

resistance, on one hand, and reduced insulin sensitivity, on the other hand (Caprio et al. 1989;

Cook et al. 1993). Thus, puberty is a crucial time for the development and diagnosis of IRS.

It has been reported that the pathogenesis of IRS appears to involve a defective

intracellular insulin signalling pathway, primarily in hepatocytes (Kohen et al. 2003). It is

very difficult to diagnose IRS and treat at an early stage, as early molecular adaptations in

glucose metabolic, insulin signalling and leptin signalling pathways culminate into

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irrevocable clinical manifestations, only later in life. These pathways play a crucial role in

mediating the final metabolic outcomes and hence are addressed in the present study.

Pharmacological agents that restore these alterations can be useful in management of early

onset of IRS.

The physiological development of Rattus norvegicus, has been chronicled as- infancy

(3rd

-4th

wk of age), childhood (4th

-6th

wk), peri-pubertal (6th

-8th

wk), adolescence (8th

-10th

wk) and adulthood (10th

wk onwards) (Sengupta 2013). Thus, in the present study design, the

4th

-6th

week corresponds to peri-pubertal and adolescent stage of animals, and 6th

-8th

week

corresponds to young adulthood. Drinking fructose (15%) was provided to rats over 8 weeks

ie, spanning from pre-pubertal, pubertal and post-pubertal stages.

Present study revealed that the food and fructose intake in NC steadily rose up to 4-5th

week of study, i.e., up to puberty, and plateaus off thereafter, i.e, post pubertal-adulthood

stage. The food intake in AMT and FC follows a similar pattern; with food intake

significantly lower in former than latter. It was also observed that chow feed intake was

reduced in AMT as compared to NC. Through series of taste aversion tests, AM could not be

implicated with the same (data not shown here). It may be hypothesized and corroborated

through further studies that, AM may be involved in re-setting of central mechanism(s) that

control food intake and satiety. A sharp rise in FBG was recorded at puberty and the trend of

elevated FBG continued thereafter in FC but not in NC and AMT. Thus, the food choices at

peri-puberty initiated onset of IRS at young adulthood that may be irretrievable

The present study explores the effect of aqueous leaf extract of Aegle marmelos (500

mg/kg/day), in conferring protection against the metabolic correlates developed as a result of

excess fructose intake in the developmental stages in rodents. The aqueous leaf extract of

Aegle marmelos is reported to contain flavonoids in high concentrations (Siddique et al.

2010). Rutin, a flavanol, is reported to be present in A.marmelos (Patil et al. 2015) and was

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identified as PRS in the present study to authenticate AM. In keeping with the guidelines

elucidated by global bodies regarding authentication of plant extracts before their

pharmacological assessment, the rutin content in AM was estimated to be 8.52 µg/mg of AM.

It is well known that degree of sweetness accounts for greater palatability and that the

desire for calorically sweetened solution reduces the intake of solid food, but not enough to

prevent a positive caloric balance (Sclafani 1991). Data indicate that energy from beverages

generally does not displace or decrease energy from other foods consumed, leading to energy

imbalances (Wharton et al. 2004). Present study shows an increased consumption of fructose

by FC, which could be due to the preferential liking for sweet taste. Consequently animals

derived almost 30% of their total energy (calories) from fructose, although there was no

significant difference in the body weight of the animals. AM reduced the fructose

consumption to successfully restore the preference for chow diet. These findings are also in

accordance with the previously reported results which show that after chronic fructose intake,

food intake is suppressed in humans (Le et al. 2006). It can be concluded that increased body

weight is not the primary marker for development of IRS, but other metabolic alterations play

a crucial role in disease genesis, progression and precipitation.

Various reported data concludes that excess glucose produced as a result of rapid

fructose metabolism stimulates insulin release but the fructose induced insulin resistance

prevents the insulin from effectively metabolizing glucose and impairs glucose uptake along

with changes in hepatic glucose metabolism, resulting in altered metabolic milieu (Bezerra et

al. 2000). The metabolic misbalance in IRS is better diagnosed by level of glucose

intolerance rather than fasting blood glucose measures (Phillips et al. 2006). In our data a

significant change in the fasting blood glucose concentration over 8 weeks in FC as compared

to NC was not recorded, that is also in concordance with the negligible body weight changes

in FC. The FBG values for AMT were also close to NC and FC overall, with significant

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changes only at 5th

and 7th

week compared to FC. Previous studies report an increase of 30%

in AUC in fructose control group when drinking water was replaced with 10% fructose

solution (Rebollo et al. 2014). In the present study, data from OGTT experiments show that

the blood glucose concentration reached maxima within 15 min of oral glucose load in both

FC and NC, but the concentration in former was significantly higher than the latter (p<0.05).

Further, typical second peak at 60 min was recorded in NC but not FC, with a 17% increased

AUC in FC, suggesting a state of altered glucose absorption, insulin response and glucose

tolerance. Thus, it may be summarized that increased hepatic availability of fructose leads to

production of excess glucose and glycogen via gluconeogenesis, on one hand, and hindered

response to insulin, on the other, to collectively cause tissue glucose intolerance. AM reduced

the FBG levels drastically after 5th

week of the study as compared to FC. Also a significant

reduction in blood glucose levels were recorded at 15 and 30 min time points in OGTT. Our

data is in agreement with the previous studies reporting that Aegle marmelos, 250 mg/kg

leads to lowering of average blood glucose levels and significantly improves the glucose

tolerance curve (Sachdewa et al. 2001).

It is evident that hepatic metabolism of fructose favors de novo lipogenesis (Kok et al.

1996) and decreased triglyceride clearance from the body that is linked with hyperlipidemia

and increased body fat stores. We also report a state of dyslipidemia with marked

hypertriglyceridemia, with increased liver weight and intra-hepatic infiltration of triglycerides

in animals consuming fructose. Treatment with AM significantly reduced the TG, TC, VLDL

and LDL levels along with a significant reduction in liver weight as compared to FC,

suggesting its potential as a hypolipidemic agent.

Here we report the decreased activity of hepatic hexokinase enzyme and increased

levels of liver glucose-6-phosphatse and fructose-1,6-bisphosphatase in FC indicating

decreased peripheral glucose utilization and activation of gluconeogenic pathway

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respectively. Rebolloa and co-workers found increased activity of liver gluconeogenic

enzymes in animals on fructose diet (10%) for 2 weeks (Rebolloa et al. 2014). Previous

studies report that fructose-1,6-bisphosphatase and glucose-6-phosphatase induction is

consistent with robust accumulation of glycogen in fructose-fed rats (Hyung et al. 2008). AM

reinforced the glycolytic pathway over gluconeogenic pathway and reduced glycogen levels

in the liver to meet the energy requirements of the body. Studies have shown that treatment

with Aegle marmelos increased hexokinase activity, indicating an overall increase in glucose

influx (Sharma et al. 2007).

Several literature reports indicate that leptin resistance characterized by

hyperleptinemia is a component of IRS (Suzuki et al, 2004). Present study also confirms a

state of systemic hyperleptinemia in FC (∼14X vs NC), which was irrespective of increased

body weight. Treatment with AM for 8 weeks decreased the plasma insulin (∼3.5X) and

leptin (∼12X) levels, with increased downstream sensitivity as confirmed by upregulated

PI3K/AKT and JAK-STAT 3 pathways, respectively.

CONCLUSION

Liver plays a central role in regulation of the metabolic input and output of the body

and is exquisitely sensitive to changes in metabolism of nutrients like fructose. Over

expression of GLUT 2 receptors on the cellular membrane of hepatocytes of FC as seen in the

immunohistochemistry confirms excess uptake of fructose by the hepatocytes. Excessive

fructose causes a reversal of the glycolytic pathway and leads to production of glucose via

gluconeogenetic pathway. Although a high quantum of insulin is released in response to the

increased levels of glucose, but it is not translated into downstream signalling. A feeble

response to hyperinsulinemic state is elicited that creates chronic hyperglycaemic state

throughout critical developmental stages of pre-puberty and puberty to ultimately manifest as

IRS at adulthood.

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Aqueous leaf extract of Aegle marmelos (500 mg/kg) beneficially stimulated the

downstream insulin and leptin cascade to impede development of IRS.

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Table 1: Effect of AM (500 mg/kg, po) on lipid profile, visceral weight, levels of plasma

insulin and leptin, and liver glycogen content of rats provided drinking fructose

from weaning to adulthood.

NC FC AMT

TG(mmol/l) 125.5±23.50 156.5±2.50a 106.5±1.50

c

TC(mmol/l) 170.5±15.50 191.5±3.50 130±1c

LDL(mmol/l) 99.9±11.30 117.7±2.50 61.7±0.30c

HDL(mmol/l) 45.7±0.25 42.5±1.5 47±1c

VLDL(mmol/l) 25.1±4.7 31.3±0.501a 21.3±0.30

c

Wt of Liver (g) 7.903±0.183 8.656±0.144a 6.35±0.218

b

Wt of Kidney (g) 1±0.031 1.07±0.043a 0.84±0.024

b

Wt of Heart (g) 1.05±0.024 0.98±0.022a 0.83±0.051

b

Wt of Intestine (g) 7.55±0.30 8.08±0.30a 6.54±0.395

b

Plasma Insulin

(ng/ml) 0.432±0.103 1.406±0.41

d 0.43±0.13

b

Plasma Leptin

(pg/ml) 110.6±37.55 595.66±198

d 42.57±6.06

b

HOMA-IR 2.41±0.60 8.45±2.52d 2.53±0.75

b

Glycogen (µg/gm

liver) 5.7229+ 0.0258 7.8414+0.181

a 5.507+0.024

c

Each value is the mean+SEM of six rats. a p<0.05 vs NC, b p<0.001 vs FC, c p<0.05 vs FC, d

p<0.001 vs NC

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LEGENDS

Fig 1: RP-HPLC Chromatogram of aqueous extract of leaves of Aegle marmelos (500

µg/ml) (a) and standard rutin (40 µg/ml) (b). Well separated, finely resolved, sharp peak of

Rutin was detected at 256 nm in extract and standard eluting at Rt=7.12 ±0.04 min using

Methanol:Water (1:1), pH 2.8, at flow rate of 1 ml/min.

Fig 2: Measurement of weekly food intake (a); fructose intake (b); body weight (c); fasting

blood sugar (d) and OGTT-AUC (e) of animals in NC, FC and AMT. The food and fructose

intake in NC steadily rises up to 4-5th

week of study, i.e., up to puberty, and plateaus off

thereafter, i.e, post pubertal-adulthood stage. The food intake in AMT and FC follows a

similar pattern; with food intake significantly lower in former than latter. The weight gain

pattern is parallel in all the three groups with lowest gain in AMT. A sharp rise in FBG was

recorded at puberty and the trend of elevated FBG continued thereafter in FC but not in NC

and AMT. At the study end, OGTT evidenced a state of glucose intolerance in FC but not in

NC and AMT. All values are mean±SEM; (n=6), a p<0.05 vs NC, b p<0.001 vs FC, c p<0.05

vs FC, d p<0.001 vs NC.

Fig 3: Effect of AM (500 mg/kg, po) on levels of hepatic enzyme and insulin and leptin

downstream signalling transducers. Relative level of hexokinase (glycolytic enzyme) was

reduced in FC but was restored by AM (a). Gluconeogenesis enzyme viz., Glucose 6-

phosphatase (b) and Fructose 1,6 biphosphatase (c) were significantly elevated in FC but

reduced by AM. Classical condition of leptin downstream resistance was evident in FC but

not AMT as the relative level of JAK STAT3 was significantly lower in former despite

fourteen times higher plasma leptin levels (e). Similarly, insulin downstream resistance was

recorded in FC but not AMT as levels of PI3-AkT were significantly lower in former but not

latter despite more than three times plasma insulin (f). Each value is the mean+SEM of six

rats. a p<0.05 vs NC, b p<0.001 vs FC, c p<0.05 vs FC, d p<0.001 vs NC.

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Fig 4 : Photomicrograph of liver parenchyma sections showing normal liver and hepatocyte

trabeculae in NC (a); macrovesicular fatty changes in FC (b); minimal fat accumulation in

hepatocyte in AMT (H and EX200) (c). Photomicrograph (200X) of immunohistochemical

staining for GLUT 2 protein in liver sections showing patchy appearance in NC (d); but

abundant expression in FC (e) that was reduced in AMT (f).

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Fig 1: RP-HPLC Chromatogram of aqueous extract of leaves of Aegle marmelos (500 µg/ml) (a) and standard rutin (40 µg/ml) (b). Well separated, finely resolved, sharp peak of Rutin was detected at 256 nm in extract and standard eluting at Rt=7.12 ±0.04 min using Methanol:Water (1:1), pH 2.8, at flow rate of 1

ml/min.

81x60mm (300 x 300 DPI)

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Fig 2: Measurement of weekly food intake (a); fructose intake (b); body weight (c); fasting blood sugar (d) and OGTT-AUC (e) of animals in NC, FC and AMT. The food and fructose intake in NC steadily rises up to 4-5th week of study, i.e., up to puberty, and plateaus off thereafter, i.e, post pubertal-adulthood stage. The

food intake in AMT and FC follows a similar pattern; with food intake significantly lower in former than latter. The weight gain pattern is parallel in all the three groups with lowest gain in AMT. A sharp rise in FBG was recorded at puberty and the trend of elevated FBG continued thereafter in FC but not in NC and AMT. At the

study end, OGTT evidenced a state of glucose intolerance in FC but not in NC and AMT. All values are mean±SEM; (n=6), a p<0.05 vs NC, b p<0.001 vs FC, c p<0.05 vs FC, d p<0.001 vs NC.

81x60mm (300 x 300 DPI)

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Fig 3: Effect of AM (500 mg/kg, po) on levels of hepatic enzyme and insulin and leptin downstream signalling transducers. Relative level of hexokinase (glycolytic enzyme) was reduced in FC but was restored by AM (a). Gluconeogenesis enzyme viz., Glucose 6-phosphatase (b) and Fructose 1,6 biphosphatase (c)

were significantly elevated in FC but reduced by AM. Classical condition of leptin downstream resistance was evident in FC but not AMT as the relative level of JAK STAT3 was significantly lower in former despite

fourteen times higher plasma leptin levels (e). Similarly, insulin downstream resistance was recorded in FC but not AMT as levels of PI3-AkT were significantly lower in former but not latter despite more than three times plasma insulin (f). Each value is the mean+SEM of six rats. a p<0.05 vs NC, b p<0.001 vs FC, c

p<0.05 vs FC, d p<0.001 vs NC.

81x60mm (300 x 300 DPI)

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Fig 4 : Photomicrograph of liver parenchyma sections showing normal liver and hepatocyte trabeculae in NC (a); macrovesicular fatty changes in FC (b); minimal fat accumulation in hepatocyte in AMT (H and EX200) (c). Photomicrograph (200X) of immunohistochemical staining for GLUT 2 protein in liver sections showing

patchy appearance in NC (d); but abundant expression in FC (e) that was reduced in AMT (f).

81x60mm (300 x 300 DPI)

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Aegle marmelos (L.) Corr. impedes onset of Insulin ... · Draft 1 Aegle marmelos (L.) Corr. impedes onset of Insulin resistance syndrome in rats provided with drinking fructose from