Curcumin pretreatment mediates antidiabetogenesis via functional regulation of adrenergic receptor subtypes in the pancreas of multiple low-dose streptozotocin-induced diabetic rats

    Curcumin pre-treatment mediates anti-diabetogenesis via functional regulation of adrenergic receptor subtypes in the pancreas of MLD-STZ induced diabetic rats George Naijil, T.R. Anju, S. Jayanarayanan, C.S. Paulose PII: DOI: Reference:

S0271-5317(15)00160-8 doi: 10.1016/j.nutres.2015.06.011 NTR 7505

To appear in:

Nutrition Research

Received date: Revised date: Accepted date:

3 January 2015 15 June 2015 30 June 2015

Please cite this article as: Naijil George, Anju TR, Jayanarayanan S, Paulose CS, Curcumin pre-treatment mediates anti-diabetogenesis via functional regulation of adrenergic receptor subtypes in the pancreas of MLD-STZ induced diabetic rats, Nutrition Research (2015), doi: 10.1016/j.nutres.2015.06.011

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ACCEPTED MANUSCRIPT Curcumin pre-treatment mediates anti-diabetogenesis via functional regulation of

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Naijil George, Anju T. R., Jayanarayanan S. & Paulose C. S. *

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adrenergic receptor subtypes in the pancreas of MLD-STZ induced diabetic rats

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Molecular Neurobiology and Cell Biology Unit, Centre for Neuroscience, Department of

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*Name and Address of Corresponding Author

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Biotechnology, Cochin University of Science and Technology, Cochin-682 022, Kerala, INDIA.

Dr. C.S. Paulose, Molecular Neurobiology and Cell Biology Unit,

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Centre for Neuroscience, Department of Biotechnology,

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Cochin University of Science and Technology, Cochin-682 022, Kerala, INDIA.

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Tel: +91 484 2575588,

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+91 484 2576267

Fax: + 91 484 2575588, +91 484 2576699 Email: [email protected] [email protected]

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List of abbreviations

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Streptozotocin

MLD-STZ

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Multiple low dose streptozotocin

CREB

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cAMP response element-binding protein

GLUT

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Glucose transporter

PBST

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Phosphate buffered saline with Triton X- 100

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STZ

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ACCEPTED MANUSCRIPT Abstract Lifestyle modification pivoting on nutritional management holds tremendous potential to

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meet the challenge of management of diabetes. The current study hypothesizes that regular uptake of curcumin lowers the incidence of diabetes by functional regulation of pancreatic

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adrenergic receptor subtypes. The specific objective of the study was to identify the regulatory pathways implicated in the anti-diabetogenesis effect of curcumin in multiple low dose

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streptozotocin (MLD-STZ) induced diabetic Wistar rats. Administration of MLD-STZ to

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curcumin pre-treated rats induced a prediabetic condition. Scatchard analysis, Real Time PCR and confocal microscopic studies confirmed a significant increase in α2 adrenergic receptor

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expression in the pancreas of diabetic rats. Pre-treatment with curcumin significantly decreased α2 adrenergic receptor expression. The diabetic group showed a significant decrease in the

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expression of β adrenergic receptors when compared to control. Pre-treatment significantly

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increased β adrenergic receptor expression to near control. When compared to the diabetic rats, a significant up regulation of CREB, phospholipase C, insulin receptor and GLUT 2 were

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observed in the pre-treated group. Curcumin pre-treatment was also able to maintain near control levels of cAMP, cGMP and IP3. These results indicate that marked decline in α2 adrenergic receptor function relents sympathetic inhibition of insulin release. It also follows that escalated signaling through β adrenergic receptors mediate neuronal stimulation of hyperglycemia induced beta cell compensatory response. Curcumin mediated functional regulation of adrenergic receptors and modulation of key cell signaling molecules improves pancreatic glucose sensing, insulin gene expression and insulin secretion.

Key Words: - Curcumin, Diabetes, Adrenergic receptor, Pancreas, beta cell 3

ACCEPTED MANUSCRIPT 1. Introduction

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The term "diabetes mellitus" describes a metabolic disorder of multiple aetiology,

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characterized by chronic hyperglycaemia with disturbances of carbohydrate, fat and protein metabolism, resulting from defects in insulin secretion, insulin action, or both. The effects of

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diabetes mellitus include long-term damage, dysfunction and failure of various organs. 382

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million people worldwide, or 8.3% of adults, were estimated to have diabetes in 2013. According to International Diabetes Federation, 175 million people with diabetes remain undiagnosed on a

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global scale [1]. Diabetes mellitus is associated with severe medical complications and these impose a heavy economic burden on the affected. This drew our attention on its preventive

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strategies. Diet and nutrition that commences from birth and even prior to birth play an important

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role in the development of diabetes [2]. Gaining insight into the role of nutraceuticals that can

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delay or prevent diabetes will be conducive to the development of potent novel therapeutics.

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Turmeric (Curcuma longa), a rhizomatous monocotyledonous perennial herbaceous plant of the ginger family (Zingiberaceae), has been used for the treatment of diabetes in Ayurvedic and traditional Chinese medicine [3]. Curcumin [1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6heptadiene-3,5-dione] is the major active component of turmeric. It has been shown to exhibit a wide range of pharmacological activities including anti-inflammatory, anti-cancer, anti-oxidant, anti-atherosclerotic, anti-microbial and wound healing effects [4]. Curcumin has been demonstrated to delay the development of diabetes by improving beta cell function, preventing beta cell death and by reducing pancreatic oxidative stress, inflammation, and insulin resistance in animal models [5-7].

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Insulin output from pancreatic beta cells depends on signals from endocrine, neural and

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metabolic origins [8]. Among the regulatory signals, neurotransmitters generated by the central

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nervous system play a considerable role. The pancreatic islets are richly innervated by sympathetic and parasympathetic branches of the autonomic nervous system [9]. Norepinephrine

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is the major neurotransmitter released at the sympathetic nerve terminals of pancreatic islets [10].

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Activation or inhibition of the sympathetic nervous system is under the control of glucoseexcited or glucose-inhibited neurons located at different anatomical sites, mainly in the brain

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stem and hippocampus [11, 12]. Activation of these neurons by hyperglycemia or hypoglycemia

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is a pivotal point for the control of glucose homeostasis, islet function and insulin release [13].

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The central hypothesis of the current study is that the regular uptake of curcumin lowers the incidence of diabetes by functional regulation of adrenergic receptor subtypes in the

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pancreas. Our objective was to identify the sympathetic pathways implicated in contributing to

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the anti-diabetogenesis effect of curcumin. This will help in devising better life style management strategies to delay or prevent diabetes. MLD-STZ induced diabetic rat models were used to evaluate the protective effects of curcumin pre-treatment. The initial phase of the study was directed to confirm the effects of curcumin in preventing or delaying the onset of diabetes. This was accomplished by studying the blood glucose and insulin levels in the pre-treated and diabetic groups. Further, sympathetic regulation of pancreatic insulin release was elucidated by studying the receptor number, binding affinity of total adrenergic, α adrenergic and β adrenergic receptor subtypes in experimental rats. The mRNA level and protein level expression of adrenergic receptor subtypes were evaluated using Real Time PCR and Immunohistochemistry

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ACCEPTED MANUSCRIPT studies using confocal microscope. Cell signaling alterations in the pancreas and brain regions associated with diabetogenesis and anti-diabetogenesis were assessed by examining the gene

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expression profiles of CREB, phospholipase C, insulin receptor and GLUT. Cell signaling

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pathways implicated in the anti-diabetogenesis effect of curcumin were studied by examining the expression of second messengers, cAMP, cGMP and IP3. This study will establish the anti-

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diabetogenesis activity of curcumin pre-treatment and will attempt to understand the cellular,

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molecular and neuronal control mechanisms in the onset of diabetes. This will pave way for the

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development of new and improved therapeutics.

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2. Methods and materials

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2.1. Materials

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Biochemicals used in the present study were purchased from Sigma Chemical Co., St.

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Louis, USA. Real-time-PCR Taqman probe assays on demand were purchased from Applied Biosystems, Foster City, CA, USA. Levo-[N-methyl-3H] Epinephrine (Sp. activity 68.6 Ci/mmol) was purchased from NEN Life Sciences products Inc., Boston, USA. [o-methyl-3H] Yohimbine (Sp. activity 83.0 Ci/mmol), DL-[4-3H] Propranolol (Sp. activity 29.0 Ci/mmol), [3H] thymidine (Sp. activity 18.0 Ci/mmol) and [3H] leucine (Sp. activity 63.0 Ci/mmol) were obtained from Amersham Life science, Buckinghamshire, UK. [3H] cAMP, [3H] cGMP and [3H] IP3 kits were purchased from American Radiolabeled Chemicals, St. Louis, USA. Radioimmunoassay kit for insulin was purchased from Baba Atomic Research Centre (BARC), Mumbai, India. All other reagents of analytical grade were purchased locally.

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2.2. Animals and Experimental design

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Male Wistar rats of 90–110 g body weight were used for all experiments. They were housed in separate cages under 12 h light and 12 h dark periods. Rats had free access to standard

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food and water ad libitum. All animal care procedures were done in accordance with the

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Institutional and National Institute of Health guidelines. All efforts were made to minimize the number of animals used and their suffering. Animals were divided into the following groups: i.

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control (C) ii. diabetic rats with no curcumin pre-treatment (D) iii. curcumin pretreated diabetic rats (D + C). Each group consisted of 6–8 animals. Curcumin pre-treated groups received oral

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supplementation of 7.5 mg/kg body weight curcumin at 24 h intervals for 60 days. This dosage

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was used in accordance with the earlier studies that showed this dosage to be optimal to induce physiological response in diabetic animal models [14 - 16]. Curcumin suspended in 0.5% sodium

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carboxymethylcellulose was administered at a constant volume of 5 ml/kg body weight [17].

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Diabetes was induced in the diabetic and curcumin pre-treated group of rats by intra-peritoneal injection of multiple low dose STZ (MLD-STZ) for five consecutive days [18]. STZ was freshly dissolved in 0.1 M citrate buffer, pH 4.5. Control rats were injected with citrate buffer alone. Diabetes was confirmed on the 14th day by checking the fasting blood sugar and plasma insulin levels. Rats were euthanized on 15th day by decapitation using Animal Guillotine. The pancreas was dissected out quickly over ice and the tissues collected were stored at −80°C until assay.

2.3. Estimation of blood glucose and circulating insulin levels

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ACCEPTED MANUSCRIPT Blood samples were collected from the tail vein and glucose levels were estimated by spectrophotometric method using glucose oxidase–peroxidase reactions. Insulin assay was done

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according to the procedure of BARC radioimmunoassay kit. The radioimmunoassay method is

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based on the competition between unlabeled insulin and [125I] insulin for limited binding sites on a specific antibody. At the end of incubation, antibody bound and free insulin were separated by

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the second antibody-polyethylene glycol aided separation method. Radioactivity associated with

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the bound fraction of sample and standards was measured to quantitate insulin concentration of

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samples [19].

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2.4. [3H] Thymidine and [3H] Leucine incorporation studies

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Fifteen days after the beginning of MLD-STZ treatment, pancreatic islets were isolated from all the experimental groups by standard collagenase digestion procedure employing aseptic

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techniques [20]. The islets were isolated in HEPES-buffered sodium free Hanks Balanced Salt

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Solution (HBSS) [21]. Trypan blue exclusion assay was used to assess the viability of pancreatic islet preparation and cells with a viability of >90% were chosen for all experiments. 150 µl of pancreatic beta cell suspension (cell density of 1.6 × 105 cells/cm2) was /added to poly L-lysine coated glass slide. For all experimental groups, [3H] leucine was added to one set of culture plates to determine the extent of protein synthesis and [3H] thymidine was added to the next set to determine the extent of DNA synthesis. The cells were incubated for 24 h at 37 °C in 5% CO2 atmosphere. The cells were scrapped off from the culture plates and centrifuged at 2000 x g for 20 minutes. The supernatant was discarded and the pellet was resuspended in 50 µl, 1M NaOH

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ACCEPTED MANUSCRIPT and kept overnight. Bound radioactivity was counted with cocktail-T in a Perkin Elmer Tri-Carb

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2810 TR liquid scintillation analyzer.

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2.5. Total adrenergic, α2 adrenergic and β adrenergic receptor binding studies

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[3H] Epinephrine, [3H] Yohimbine and [3H] Propranolol binding assays in pancreas were

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done according to the modified procedure of U’Prichard and Snyder [22]. The pancreas was homogenized in Tris-HCl buffer and centrifuged. The pellet was resuspended in an appropriate

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volume of incubation buffer and binding assay was performed using different radio ligand concentrations. [3H] epinephrine, [3H] yohimbine and [3H] propranolol were used for total

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adrenergic receptor assay, α2 adrenergic receptor assay and β adrenergic receptor assays,

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respectively. Non-specific binding was determined using 100 µM unlabeled epinephrine, phentolamine and propranolol for total adrenergic receptor, α2 adrenergic receptor and β

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adrenergic receptors, respectively. The reaction volume of 250 µl contained 200-250 µg protein.

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Tubes were incubated at 37 °C for 15 minutes. After incubation, the mixture was filtered rapidly through GF/B filters (Whatman) for total adrenergic receptor assay and GF/C filters (Whatman) for α2 adrenergic receptor and β adrenergic receptor binding studies. The filters were washed thrice rapidly with 5 ml of ice cold 50 mM Tris-HCl buffer, pH 7.7. Bound radioactivity was counted with cocktail-T in a Perkin Elmer Tri-Carb 2810 TR liquid scintillation analyzer.

2.6. Analysis of gene expression by Real-time PCR

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ACCEPTED MANUSCRIPT RNA was isolated from the pancreas of experimental rats using the Tri-reagent according to the procedure of Chomczynski and Sacchi [23]. Total cDNA synthesis was performed using

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ABI PRISM cDNA archive kit in 0.2 ml microfuge tubes. The reaction mixture of 20 μl

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contained 0.2 μg total RNA, 10× RT buffer, 25× dNTP mixture, 10× random primers, MultiScribe RT (50 U/μl) and RNase free water. The cDNA synthesis reactions were carried out

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at 25 °C for 10 minutes and 37 °C for 2 h using an Eppendorf Personal Cycler. Real-time-PCR

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assays were performed in 96-well plates in ABI 7300 Real-time-PCR instrument (Applied Biosystems, Foster City, USA). The specific primers and probes were purchased from Applied

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Biosystems. The TaqMan reaction mixture of 20 μl contained 25 ng of total RNA-derived cDNA, 200 nM each of forward primer and reverse primer, TaqMan probe for assay on demand,

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endogenous control β-actin and 12.5 μl of Taqman 2× Universal PCR Master Mix (Applied

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Biosystems) and RNAse free water. The following thermal cycling profile was used; 50 °C for 2 minutes, 95 °C for 10 minutes and 40 cycles of 95 °C for 15 s and 60 °C for 1 minute.

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Fluorescence signals measured during amplification were considered positive if the fluorescence

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intensity was 20-fold greater than the standard deviation of the baseline fluorescence. The ΔΔCT method of relative quantification was used to determine the fold change in expression. This was done by normalizing the resulting threshold cycle (CT) values of the target mRNAs to the CT values of the internal control β-actin in the same samples (ΔCT=CT Target – CT β-actin). It was further normalized with the control (ΔΔCT = ΔCT − CT Control). The fold change in expression was then obtained as (2−ΔΔCT) and graph was plotted using log 2−ΔΔCT [24].

2.7. Immunohistochemistry of α2 adrenergic and β adrenergic receptors in the pancreas of experimental rats using confocal microscope

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The experimental rats were deeply anesthetized and trancardially perfused with

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Phosphate buffered saline (pH 7.4) followed by 4% paraformaldehyde in PBS [25]. After

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perfusion, pancreas from each experimental group was dissected out and fixed in 4% paraformaldehyde for 1 h and equilibrated with 30% sucrose solution in PBS. 10 μm sections of

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pancreas were cut using Cryostat (Leica, CM1510 S). The sections were washed with PBS and

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then blocked with PBS with Triton X- 100 (PBST) containing 5% normal goat serum for 1 h. The primary antibodies of α2 adrenergic receptor and β adrenergic receptor (1:400 dilution in

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PBST with 5% normal goat serum) were added to the respective sections and incubated overnight at 4 °C. After overnight incubation, the tissue slices were rinsed with PBS and

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incubated with fluorescent labeled secondary antibody, prepared in PBST with 5% normal goat

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serum at 1:1000 dilution for 2 h at room temperature. The sections were observed and photographed using confocal imaging system (Leica TCS SP5 laser scanning confocal

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microscope). Quantification was done using Leica Application Suite Advanced Fluorescence

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(LASAF) software by considering the mean pixel intensity of the image. The fluorescence obtained depends on the number of receptors specific to the added primary antibody. All the imaging parameters in the confocal imaging system like photomultiplier tube (PMT), pinhole and zoom factor were kept constant for imaging the sections of all experimental groups [26].

2.8. Second messenger assay

Pancreas of the experimental rats were homogenized in a polytron homogenizer with cold 50 mM Tris-HCl buffer, pH 7.4, containing 1 mM EDTA to obtain a 15% homogenate. The

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ACCEPTED MANUSCRIPT homogenate was centrifuged at 30,000 x g for 30 minutes and the supernatant was transferred to fresh tubes for IP3, cAMP and cGMP assays using [3H]IP3, [3H]cAMP and [3H]cGMP Biotrak

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assay system kits [19, 24]. The unknown concentrations were determined from the standard

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curve using appropriate dilutions.

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2.9. Statistical Analyses

Data are presented as means with their standard errors (means ± SEM). Statistical

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evaluations were done by ANOVA using InStat (Ver.2.04a) computer program. Student– Newman–Keuls post hoc test was used to compare data among the groups. Linear regression

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Scatchard plots were made using Sigma Plot software (version 2.0, Jandel GmbH, Erkrath,

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Germany). Competitive binding data were analyzed using non-linear regression curve fitting procedure (GraphPad PRISMTM, San Diego, USA). Relative Quantification Software was used

3. Results

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to analyze Real-Time PCR results.

3.1. Fasting blood glucose level and circulating insulin level on final day (Day 14) after MLDSTZ injection in experimental rats

Fourteen days after MLD-STZ administration, the diabetic group showed a significant (p<0.001) increase in fasting blood glucose level when compared to control. Pre-treated rats (C + D) showed a significant (p<0.01) increase in blood glucose compared to control. However,

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ACCEPTED MANUSCRIPT fasting blood glucose levels of rats pre-treated with curcumin was significantly (p<0.001)

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decreased when compared to the diabetic group. (Table-1)

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Circulating insulin level was significantly (p<0.001) decreased in diabetic group when compared to control. Pre-treatment with curcumin significantly (p<0.01) averted the decrease in

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plasma insulin level. (Table-1)

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3.2. [3H] Thymidine and [3H] Leucine incorporation studies

Pancreatic beta cells isolated from diabetic rats showed a significant decrease in [ 3H]

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thymidine (p<0.05) and [3H] leucine (p<0.01) incorporation when compared to control. Beta

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cells from curcumin pre-treated rats showed a significant (p<0.001) increase in [3H] thymidine

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and [3H] Leucine incorporation when compared to both control and diabetic groups. (Table- 2)

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3.3. Scatchard analysis of total adrenergic receptor using [3H] epinephrine binding against epinephrine in the pancreas of experimental rats

Scatchard analysis of total adrenergic receptor using [3H] epinephrine binding against epinephrine in the pancreas of diabetic rats showed a significant (p<0.01) increase in Bmax when compared to control rats. Pre-treatment with curcumin significantly (p<0.01) retained the Bmax to near control when compared to diabetic group. There was no significant change in Kd in all the experimental groups. (Table- 3)

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ACCEPTED MANUSCRIPT 3.4. α2 adrenergic receptor expression in the pancreas of experimental rats

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Bmax of α2 adrenergic receptors was significantly increased (p<0.001) in diabetic group

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when compared to control. Pre-treatment with curcumin (p<0.001) significantly decreased Bmax when compared to the diabetic group. In receptor studies, there was no significant change in the

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Kd values of experimental rats (Table- 2). Gene expression studies using Real Time PCR and

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immunohistochemical studies confirmed that α2 adrenergic receptor mRNA and protein expressions were significantly up regulated in the diabetic (p<0.01) group when compared to

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control. When compared to the diabetic group, α2 adrenergic receptor expression was

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significantly (p<0.01) decreased in the C + D group. (Table- 4 & 5, Figure- 1)

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3.5. β adrenergic receptor expression in the pancreas of experimental rats

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In the diabetic group, Bmax and Kd of β adrenergic receptors were significantly (p<0.01)

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decreased when compared to control. Rats pre-treated with curcumin showed a significant increase in Bmax and Kd when compared to control (p<0.05) and diabetic (p<0.001) rats (Table2). mRNA and protein level expression of β2 adrenergic receptors showed a significant down regulation (p<0.001) in the pancreas of diabetic rats when compared to control. Pre-treatment withwith curcumin significantly (p<0.001) up regulated the β2 adrenergic receptor gene expression when compared to the diabetic group. (Table- 4 & 5, Figure- 2)

3.6. Real Time PCR amplification of CREB mRNA in the pancreas of experimental rats

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ACCEPTED MANUSCRIPT Gene expression of CREB mRNA showed a significant (p<0.001) down regulation in the pancreas of diabetic rats when compared to control. Pre-treatment with curcumin significantly

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(p<0.001) up regulated the CREB mRNA gene expression when compared to both control and

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diabetic groups. (Table- 6)

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3.7. Real Time PCR amplification of phospholipase C mRNA in the pancreas of experimental

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rats

Gene expression studies showed that phospholipase C mRNA was significantly down

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regulated (p<0.05) in the diabetic group when compared to control. In C + D there was a significant up regulation (p<0.001) of phospholipase C expression when compared to both

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control and diabetic groups. (Table- 6)

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experimental rats

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3.8. Real Time PCR amplification of insulin receptor and GLUT 2 mRNA in the pancreas of

Real time PCR gene expression of insulin receptor and GLUT 2 showed a significant (p<0.001) down regulation in the pancreas of diabetic rats when compared to control. Curcumin pre-treated rats showed a significant (p<0.001) up regulation when compared to both control and diabetic groups. (Table- 6)

3.9. Second messenger content in the pancreas of experimental rats

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ACCEPTED MANUSCRIPT The cAMP, cGMP and IP3 content in the pancreas of diabetic rats showed a significant decrease (p<0.001) when compared to control. Pre-treatment with curcumin significantly

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(p<0.001) retained a near control level of second messengers when compared to the diabetic

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group. (Table- 7)

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4. Discussion

Nutritional management holds tremendous potential to meet the challenge of diabetes

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management. The present study substantiates the role of curcumin pre-treatment in the prevention of diabetogenesis in MLD-STZ induced diabetic rat models. Wistar rats injected with

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MLD-STZ developed diabetes mellitus, characterized by hypoinsulinemia and hyperglycaemia.

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In the curcumin pre-treated rats, only a prediabetes condition developed after fourteen days of MLD-STZ injection. Prediabetes is a transition stage between normoglycaemia and uncontrolled

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hyperglycaemia, characterized by fasting blood glucose levels between 100 to 125 mg/dl [27].

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This condition can either revert back to normoglycaemia or progress to a diabetic state. Annually, up to around 10% of prediabetic individuals regain normal glucose homeostasis. Genetic, environmental and lifestyle factors lead to the reversal or persistence of this prediabetes condition or its progress to diabetes. [28]. Our data shows that beta cells isolated from diabetic rats have significantly decreased tritiated thymidine and tritiated leucine incorporation capabilities, indicating reduced DNA and protein synthesis. This decreased proliferative capacity of pancreatic beta cells has an important role in the pathophysiology of MLD-STZ induced diabetic rat models [29]. The pretreated group

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ACCEPTED MANUSCRIPT showed a significant increase in the beta cell proliferation potential, indicative of activation of beta cell compensatory response.

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Pancreatic insulin output is regulated by nutritional status, hormonal levels and neuronal

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signaling [30]. Pancreatic beta cells resemble neurons in electrical excitability, depolarization induced secretion and expression of various neurotransmitter receptors [31]. Hence, activation of

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neurotransmitter receptors present on the beta cells directly control insulin synthesis, storage,

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secretion and beta cell mass. Earlier studies from our laboratory have elucidated the neuroprotective role of curcumin in the maintenance of glucose homeostasis, mediated by

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functional regulation of muscarinic and glutamatergic receptors in the pancreas [32, 33].The current study observed differential functional regulation of adrenergic receptor subtypes in the

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pancreas. Out of the different subtypes of α adrenergic receptors, α2 is the predominantly

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expressed one in pancreas. Radio receptor assay, Real-Time PCR analysis and confocal studies confirmed the significant increase of α2 adrenergic receptors in the pancreas of diabetic rats.

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Activation of α2 subtype of adrenergic receptors regulate different intracellular pathways to

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modulate beta cell function by coupling to different G proteins [34]. Activated α2 adrenergic receptors inhibit the activity of adenylyl cyclase and reduce intracellular cAMP levels. cAMP potentiates hyperglycaemia induced insulin secretion by activating protein kinase A and Epac [35, 36]. α2 adrenergic receptors also prevent calcium mediated exocytosis of insulin vesicles by potassium channel mediated hyperpolarization [37]. Increased potassium channel activity and suppression of calcium current deteriorates beta cell membrane depolarization induced insulin secretion [38]. Pre-treatment with curcumin significantly reverted the changes in α2 adrenergic receptor expression to near control. This may be attributed to the reduced inhibition over insulin release by the activation of adenylyl cyclase, decrease of potassium channel activity and increase

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ACCEPTED MANUSCRIPT of calcium current. Knock out of α2 adrenergic receptor in mice and administration of adrenoceptor specific antagonists to diabetic rats and humans have showed improved glucose

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tolerance and increased insulin secretion [39, 40, 41].

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In contrast to α2-adrenoceptors, pancreatic β2 adrenergic receptors showed a significant decrease in receptor binding, mRNA and protein expression levels in diabetic rats when

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compared to the control group. Curcumin pre-treatment significantly increased the overall

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expression of β2 adrenergic receptors when compared to the diabetic group. Increased β2 adrenergic receptor activates cell cycle progression and beta cell proliferation by activation of

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adenylyl cyclase and protein kinase A [42]. Activated protein kinase A promotes pancreatic regeneration by transcriptional activation of several key cell proliferation factors. [43]. β2

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adrenergic receptor action through ERK and Akt pathways inhibit apoptotic factors like Bax and

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caspase-3 and activate anti-apoptotic proteins like Bad , Bcl-2, Bcl-XL and Mcl-1 [44]. Ras activation mediated by β2 adrenergic receptor modulates the expression of several key signaling

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proteins including Raf, MEK, ERK, PI3K, Akt, PTEN and NF-κB to support beta cell

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regeneration and prevent apoptotic cell death [45, 46]. The receptor affinity showed variations in the experimental rats, which might be due to the reversible phosphorylation of β2 adrenergic receptor by protein kinases [47]. Thus, increased expression of β2 adrenergic receptor in the pancreas of pre-treated rats helps to activate beta cell compensatory response and stimulates insulin release.

Cell signaling through G protein coupled receptors and tyrosine kinase receptors control pancreatic beta cell survival, proliferation and insulin secretion by the activation of key intracellular transcription factors. Our streptozotocin induced diabetic rats showed a significant

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ACCEPTED MANUSCRIPT down regulation of CREB gene expression in pancreas. CREB on activation stimulates several cAMP response element containing proteins including those regulating glucose sensing, insulin

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gene expression, insulin secretion, beta cell survival and mass expansion [48]. Jhala et al., [49]

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reported that deficiency of functional CREB expression leads to beta cell apoptosis and subsequent development of diabetes. Diabetes associated down regulation of CREB expression is

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due to increased cytokine mediated inhibition of adenyl cyclase [50]. Curcumin pre-treatment

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significantly increased the expression of CREB and increased insulin release and proliferation of beta cells. Increased cAMP levels and CREB expression activate insulin receptor substrate 2 and

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mediate glucose responsive insulin secretion and beta cell proliferation [51]. Diabetic rats in our experiments also showed a significant decrease in phospholipase C

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expression. This results in decreased production of inositol triphosphate (IP3) and

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diacylylglycerol (DAG), crucial to the increase in intracellular Ca2+ levels and exocytosis of insulin granules [52]. Pre-treatment with curcumin resulted in significant increase in

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phospholipase C expression and the impairment of postprandial insulin secretion was reverted to

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near control. Further, diabetic group in the present study showed a significant down regulation of insulin receptor expression in the pancreas. This impairs insulin receptor mediated positive feedback system [53] and leads to the development of beta cell insulin resistance, resulting in glucose unresponsiveness [54]. Curcumin pre-treated rats showed significant up regulation of insulin receptor mRNA. This stimulates the positive feedback system to increase insulin synthesis and secretion and stimulate pathways leading to beta cell survival and proliferation [55]. MLD-STZ induced diabetic rats in our experiments showed a significant down regulation of GLUT 2 mRNA expression [56, 57]. Deficiency in functional GLUT 2 expression is associated with the development of hyperglycaemia and hypoinsulinemia [58]. Pre-treatment

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ACCEPTED MANUSCRIPT with curcumin significantly increased GLUT 2 mRNA levels, compared to the control and diabetic rats. This helps to maintain normoglycaemia after MLD-STZ administration by

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promoting normal functioning and development of beta cells.

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The present study observed a significant decrease in second messengers in the pancreas of diabetic rats. Decreased cAMP content leads to reduced insulin secretion in response to

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various insulinotropic stimuli and activates cellular apoptosis by removing the CREB mediated

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inhibition on apoptotic factors [59]. Curcumin pre-treatment was able to maintain a near control level of cAMP content. This helps to retain the sensitivity of beta cells towards various

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secretagogue stimuli. Second messenger cGMP regulates cellular functions through the activation of a specific cGMP-dependent protein kinase, protein kinase G [60]. Activation of

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protein kinase G mediated cell signaling potentiates glucose stimulated insulin secretion,

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promotes beta cell differentiation and prevents beta cell apoptosis [61]. Significant decrease in cGMP content of diabetic rat pancreas induces a disturbance in intracellular Ca2+ homeostasis

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and alters glucose stimulated insulin release [62]. Curcumin pre-treatment retained cGMP at near

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control levels. Diabetic rats showed a significant reduction in the pancreatic IP3 content, leading to decreased calcium mobilization from endoplasmic reticulum in response to glucose and other secretagogue stimuli. [63]. Curcumin pre-treated rats were able to maintain a near control level of IP3 when compared to diabetic rats. Significant increase in IP3 levels of pre-treated rats also induces an increase in cAMP levels to aid glucose mediated oscillations in insulin secretion [64].

The major strengths of the present study include its prospective design and the range of parameters that have been evaluated with respect to adrenergic receptor expression, cell signaling and second messengers. The study gives a good picture of curcumin pre-treatment

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ACCEPTED MANUSCRIPT induced functional regulation of adrenergic receptor subtypes in the pancreas of diabetic rat models. However, one limitation of the study is that dosage of curcumin used has to be

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extrapolated to the human setting, considering intestinal conjugation, bioreduction and

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pharmacological deactivation of curcumin by human intestinal tissue [65]. Once this has been explored to sufficient detail, the treatment strategy proposed in the study will have immense

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clinical value for use as a human therapeutic intervention.

Based on our results, we accept the hypothesis that regular uptake of curcumin lowers the

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incidence of diabetes by functional regulation of adrenergic receptor subtypes in the pancreas. Pre-treated rats increase beta cell glucose response by up regulation of insulinotropic β

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adrenergic receptors and down regulation of inhibitory α2 adrenergic receptors. In pre-treated

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rats, expression of intracellular signaling and glucose sensing molecules increases in response to increased metabolic demands induced by streptozotocin. In conclusion, curcumin pretreatment

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increases responsiveness of pancreas to various hyperglycemia associated insulinotropic stimuli

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by functional regulation of adrenergic receptors and associated intracellular signaling systems.

Acknowledgment

This work was supported by grants from Department of Science and technology, Department of Biotechnology, Indian Council of Medical Research, Govt. of India, and Kerala State Council for Science, Technology & Environment, Govt. of Kerala, to Dr. C. S. Paulose.

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Figure - 1

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Confocal imaging of α2 adrenergic receptor in the pancreas of experimental rats. C+D- Curcumin pre-treated diabetic rats. shows α2 adrenergic receptors. Scale bar represents 50µm.

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Figure - 2

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Confocal imaging of β2 adrenergic receptor in the pancreas of experimental rats. C+D- Curcumin pre-treated diabetic rats. shows β2 adrenergic receptors. Scale bar represents 50µm.

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β2 adrenergic receptors

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α2 adrenergic receptors

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MLD-STZ

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Curcumin Pre-treatment

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Improved Cell signaling Glucose sensing Insulin receptor and GLUT Transcription factors CREB and Phospholipase C Second messengers cAMP, cGMP and IP3

Graphical Abstract

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ACCEPTED MANUSCRIPT Captions to illustration Curcumin pre-treatment exerts anti-diabetogenetic effect via functional regulation of

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adrenergic receptor subtypes in the pancreas of multiple low dose streptozotocin (MLD-STZ)

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induced diabetic rats. Curcumin pre-treated rats increased beta cell glucose response after MLDSTZ injection by up regulation of insulinotropic β adrenergic receptors and down regulation of

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inhibitory α2 adrenergic receptors. Functional regulation of adrenergic receptor subtypes in

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pancreas modulate key cell signaling proteins (CREB and phospholipase C) and second messengers (cAMP, cGMP and IP3) and improves pancreatic glucose sensing (insulin receptor

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and GLUT), and insulin secretion in response to increased metabolic demands.

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ACCEPTED MANUSCRIPT Table-1 Fasting blood glucose and circulating insulin levels on final day (Day 14) after MLD-STZ injection in rats. Experimental groups

Diabetic

C+D

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Control 87.83 ± 2.61

305.50 ± 12.32

Insulin Concentration (μU/ml)

62.38 ± 3.91

21.90 ± 4.53

a

119.33 ± 4.65

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Blood glucose (mg/dL)

a

b,

d

50.60 ± 4.37

e

Values are means ± SEM of 4-6 separate experiments. Each group consist of 4-6 rats. C+D- Curcumin preb

p<0.01 when compared to Control.

d

p<0.001,

e

p<0.001 when compared to

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a

treated diabetic rats. p<0.001, Diabetic group.

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Diabetic

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Control

9441 ± 480

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15319 ± 460

7350 ± 318

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Values means SEM

Experimental groups 3 [ H] Thymidine incorporation (DPM/mg Protein) 3 [ H] Leucine incorporation (DPM/mg Protein)

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Table-2 [3H] Thymidine and [3H] Leucine incorporation studies in the beta cells of rats.

12377 ± 492

c

b

C+D

14272 ± 504

21493 ± 618 a

a, d

a, d

separate experiments. Each group consist of 4-6 rats. C+D- Curcumin pre-treated diabetic rats. p<0.001,

p<0.01,

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p<0.001 when compared to Diabetic group.

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p<0.05 when compared to Control.

d

b

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c

are ± of 4-6

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Table-3 Scatchard analysis of adrenergic receptor subtypes in the pancreas of rats.

Control 221.28 ± 10.23 3.03 ± 0.13

(nM)

105.45 ± 5.77

(fmoles/mg protein)

α2 adrenergic receptor

Bmax Kd (nM) a

b

c

90.60 ± 5.46

66.32 ± 3.75

4.56 ± 0.27

b

3.62 ± 0.22 d

192.75 ± 11.62

e

3.03 ± 0.16

174.37 ± 8.09 1.32 ± 0.05

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(fmoles/mg protein)

C+D

3.63 ± 0.18

1.53 ± 0.03

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Kd (nM)

β adrenergic receptor

284.83 ± 10.24

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Bmax

Diabetic

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Experimental groups Bmax (fmoles/mg Total adrenergic protein) receptor Kd

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Values are means ± S.E.M of 4-6 separate experiments. Each group consist of 4-6 rats. C+D- Curcumin pre-treated

a

112.96 ± 7.12

d

1.30 ± 0.06 b

b

116.13 ± 5.52 5.01 ± 0.25

c, d

c, d

e

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diabetic rats. p<0.001, p<0.01, p<0.05 when compared to Control. p<0.001, p<0.01 when compared to Diabetic group.

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Experimental groups α2 adrenergic receptor Log RQ β2 adrenergic receptor

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Table-4 Real Time PCR amplification of adrenergic receptor subtypes mRNA in the pancreas of rats.

Control

Diabetic

0 0

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1.02 ± 0.06

-0.37 ± 0.02

C+D

a a

0.19 ± 0.01 0.15 ± 0.01

b, d a, d

Values are means ± S.E.M of 4-6 separate experiments. Each group consist of 4-6 rats. C+D- Curcumin pre-treated a

b

d

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diabetic rats. p<0.001, p<0.01 when compared to Control. p<0.001 when compared to Diabetic group.

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Table-5 Confocal imaging of adrenergic receptor subtypes in the pancreas of rats.

Experimental groups α2 adrenergic receptor Mean Pixel Intensity β2 adrenergic receptor

Control

Diabetic

27.66 ± 1.13 36.56 ± 1.42

b

d

38.62 ± 1.32

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a

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Values are means ± S.E.M of 4-6 separate experiments. Each group consist of 4-6 rats. C+D- Curcumin pre-treated

22.91 ± 1.37

C+D b a

30.71 ± 1.29 39.27 ± 1.70

e d

e

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diabetic rats. p<0.001, p<0.01 when compared to Control. p<0.001 & p<0.01when compared to Diabetic group.

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Diabetic

-0.35 ± 0.02

Phospholipase C

0

-0.40 ± 0.03

Insulin receptor

0

GLUT 2

0

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Log RQ

Control 0

-1.56 ± 0.09 -4.40 ± 0.22

C+D a c a a

0.13 ± 0.01 2.15 ± 0.11 1.83 ± 0.10 1.04 ± 0.07

a, d a, d a, d a, d

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Experimental groups CREB

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Table-6 Real Time PCR amplification of CREB, Phospholipase C, Insulin receptor and GLUT 2 mRNA in the pancreas of rats.

Values are means ± S.E.M of 4-6 separate experiments. Each group consist of 4-6 rats. C+D- Curcumin pre-treated a

c

d

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diabetic rats. p<0.001, p<0.05 when compared to Control. p<0.001 when compared to Diabetic group.

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Control

Diabetic

16.64 ± 0.89

75.81 ± 3.81

48.22 ± 2.29

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57.95 ± 2.95

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30.43 ± 1.71

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Values means S.E.M

Experimental groups cAMP content (pmoles/mg protein) cGMP content (pmoles/mg protein) IP3 content (pmoles/mg protein)

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Table-7 Second messenger content in the pancreas of rats.

19.77 ± 1.49

a

b

a

C+D 34.33 ± 1.86

70.86 ± 3.60

49.99 ± 3.25

separate experiments. Each group consist of 4-6 rats. C+D- Curcumin pre-treated diabetic rats. d

e

a

d

e

are ± of 4-6

d

b

p<0.001, p<0.01

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when compared to Control. p<0.001, p<0.01when compared to Diabetic group.

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