Elsevier

Metabolism

Volume 65, Issue 5, May 2016, Pages 667-684
Metabolism

Basic Science
BDNF protects pancreatic β cells (RIN5F) against cytotoxic action of alloxan, streptozotocin, doxorubicin and benzo(a)pyrene in vitro

https://doi.org/10.1016/j.metabol.2016.01.016Get rights and content

Abstract

Objective

The study was conducted to observe whether brain-derived neurotrophic factor (BDNF) has cytoprotective actions against alloxan (AL), streptozotocin (STZ), doxorubicin (DB) and benzo(a)pyrene (BP) compounds in vitro that may account for its beneficial action in diabetes mellitus.

Materials and methods

This in vitro study was performed using rat insulinoma (RIN5F) cells. Possible cytoprotective action of BDNF (using pre-treatment, simultaneous and post-treatment schedules of RIN5F cells with BDNF) against the four chemicals tested was evaluated using MTT and apoptosis assays. Possible mechanism of cytoprotective action of BDNF was assessed by measuring BCl2/IKB-β/Pdx mRNA transcripts and anti-oxidant levels in RIN5F cells. Effect of alloxan, STZ, doxorubicin and BP on the production of BDNF by RIN5F cells was also studied.

Results

Results of the present study revealed that BDNF in the doses (100 ng > 50 ng > 10 ng/ml) has significant cytoprotection (P < 0.001, P < 0.01) on cytotoxic action of AL, STZ, DB and BP against rat insulinoma RIN5F (5 × 104 cells/100 μl) cells in vitro. It was observed that AL, STZ, DB and BP inhibited BDNF production significantly (P < 0.001) in a dose-dependent manner by RIN5F cells (0.5 × 106 cells/500 μl) in vitro, while BDNF not only prevented apoptosis induced by these four chemicals but also significantly increased (P < 0.001) BCl2/IKB-β/Pdx mRNA transcripts and restored anti-oxidant levels (P < 0.01) in RIN5F cells to normal.

Discussion

These results suggest that BDNF has potent cytoprotective actions, restores anti-oxidant defenses to normal and thus, prevents apoptosis and preserves insulin secreting capacity of β cells. In addition, BDNF enhanced viability of RIN 5F in vitro. Thus, BDNF not only has anti-diabetic actions but also preserves pancreatic β cells integrity and enhances their viability. These results imply that BDNF functions as an endogenous cytoprotective molecule that may explain its beneficial actions in some neurological conditions as well.

Introduction

Both obesity and type 2 diabetes mellitus are assuming epidemic proportions in developed and developing countries. Consumption of high fat diet or energy dense food and lack of exercise have been attributed to this increase in the incidence of obesity and type 2 diabetes mellitus [1], [2]. In addition, recent studies suggested that both passive and active smoking is associated with type 2 DM [3], [4]. Similarly, there is increasing evidence linking exposure to environmental toxins and susceptibility to type 2 DM [5], [6]. Air pollution (particularly particulate matter) could trigger pro-inflammatory events that predispose to the development of type 2 DM. Since air pollution is a pervasive risk factor throughout the world, even modest alleviation in exposure may provide substantial public health benefits. The MONICA/KORA Augsburg Cohort Study in which 5470 men and 5422 women (aged 25–74 years) were enrolled without diabetes who participated in surveys between 1984 and 1995 when analyzed revealed that the number of cigarettes and the nicotine and tar consumption per day were associated with a significantly increased risk of type 2 diabetes among men [7]. Previous studies showed that smoking may contribute to the development of insulin resistance [8]. Furthermore, ability of insulin to stimulate glucose uptake is significantly lower in chronic cigarette smokers [9]; smoking increases insulin resistance by increasing abdominal fat distribution and a greater waist-to-hip ratio [10] and smoking may increase free radical oxidative damage and oxidative stress [11] and it is likely that nicotine, carbon monoxide or other agents in tobacco smoke may have direct toxic effects on the pancreas and insulin receptor sensitivity [12], [13], all of which may contribute to the increased risk for type 2 DM.

One of the important components of coal tar, cigarette smoke, wood smoke, and burnt foods such as coffee and grilled goods is benzo(a)pyrene (BP) [14], one of the polycyclic aromatic hydrocarbons (PAHs). BP and related compounds intercalate into DNA, interfering with transcription and are known to be associated in the development of lung cancer. Intestinal cytochrome P450 subclass 1 A1 (CYP1A1) is needed to protect the host from ingested carcinogens such as PAH including BP. The expression of CYP1 (including CYP1A2 and CYP1B1) is dependent solely on a heterodimeric transcription factor consisting of the arylhydrocarbon receptor (AHR) and the AHR nuclear translocator (ARNT). The presence of ligands for TLR2 (toll-like receptor 2) of bacterial origin seems to be crucial for detoxication of luminal carcinogens by CYP1A1 in the intestine, indicating a complex interplay between the immune system of the host and intestinal bacteria with detoxication mechanisms [15]. It is estimated that more than 20% of the carbon in the universe may be associated with PAHs. These evidences suggest that one of the atmospheric pollutants that may have a role in the development of diabetes mellitus could be exposure to BP and other PAHs. Studies have revealed that doxorubicin (DB), a widely used anti-cancer drug, inhibits adipogenesis through the down-regulation of PPARγ and impairs blood glucose and lipid clearance that leads to the development of hyperglycemia and hyperlipidemia resulting in lipotoxicity, glucotoxicity, and inflammation and insulin resistance that are features of type 2 diabetes [16].

Brain-derived neurotrophic factor (BDNF) serves as a transmitter between immune and nervous systems and has been shown to modulate inflammation [1], [17]. Both parenteral and intrahypothalamic administration of BDNF has been shown to decrease food intake, increase energy expenditure and ameliorate hyperinsulinemia and reverse features of diabetes mellitus in experimental animals [1], [18], [19]. Plasma levels of BDNF are known to be reduced in subjects with obesity and type 2 diabetes mellitus [20], [21], though some studies do not support this contention [22]. BDNF is a neurotrophic factor that is needed for the survival of neurons and gut produces substantial amounts of BDNF [23], [24], [25], [26], [27], [28], where intestinal cytochrome P450 subclass 1 A1 (CYP1A1) is needed to protect the host from ingested carcinogens such as PAH including BP. We hypothesized that BDNF may protect pancreatic β cells from the cytotoxic action of various chemicals and thus, prevent development of diabetes mellitus. In the present study, we examined the effect of cytoprotective actions of BDNF against alloxan (AL) and streptozotocin (STZ) that are known to be toxic to β cells and BP and DB. These in vitro studies revealed that BDNF can prevent cytotoxic action of AL, STZ, BP and DB on rat pancreatic β cells (RIN5F) by restoring anti-oxidants, nitric oxide (NO) and lipid peroxides to normal; by enhancing the expression of BCL2 (anti-apoptotic) and suppressing BAX (pro-apoptotic) and inflammatory genes (NF-κB and IKB-β) and augmenting that of pancreatic homeobox (Pdx).

Section snippets

Reagents

AL, STZ and BP were purchased from Sigma Aldrich. DB was purchased from Khandelwal Laboratories (Thane, India). All culture media and additives were purchased from Sigma Aldrich Chemicals Pvt. Ltd., (Bangalore, India). BDNF and BDNF ELISA kits (CYT306) were purchased from Millipore, MA. PCR reagents were purchased from Genei, (Bangalore, India).

Cell Culture Conditions

Rat insulinoma (RIN henceforth called as RIN5F for brevity), an insulin secreting cell line obtained from the National Center for Cell Science (Pune,

Effect of AL on Viability of RIN5F Cells

AL is toxic to RIN5F by generating reactive oxygen species (ROS). To assess its cytotoxicity, cells were exposed to different concentrations of AL (1–12 mM) for different periods of time (1–3 h). It is evident from the results shown in Fig. 1A that AL has a significant (P < 0.001) and dose-dependent suppressive action on the viability of RIN5F cells. Based on these results, we used in all our subsequent studies a dose of 6 mM AL and exposure time of 1 h under which conditions only ~ 53% of the cells

Discussion

It is evident from the results of the present study that BDNF prevents AL/STZ/DB/BP-induced apoptosis of RIN5F cells in vitro that could be attributed, at least, in part, to its ability to restore the anti-oxidant status (See Table 5.) and expression of BCL2, BAX, NF-κB, IKB-β and Pdx to normal.

AL is selectively toxic to pancreatic β cells because it preferentially accumulates in these cells through uptake via the GLUT2 receptors. AL brings about its toxic action on β cells by free radicals,

Author Contributions

Conceived and designed the experiments along with idea proposal: U.N.D. Performed the experiments: S.B. Analyzed and interpreted the data: S.B. and U.N.D. Contributed to the acquisition of reagents/materials/analysis tools: U.N.D. Drafting the paper: S.B. and U.N.D. Read and approved the final manuscript: U.N.D., S.B. and S.N.

Funding

U.N.D. is in receipt of Ramalingaswami Fellowship of the Department of Biotechnology, New Delhi during the tenure of this study. B.S. is a Senior Research Fellow of Indian Council of Medical Research (ICMR), New Delhi and the work presented in this paper is part of her PhD work. This work was supported by ICMR and in part, by grants from Defence Research and Development Organisation, New Delhi ([TC/2519/INM03/2011/CARS] under R&D Project INM-311) to U.N.D. The funders had no role in study

Conflict of Interest

The authors declare that there is no conflict of interest that would prejudice the impartiality of this scientific work.

References (57)

  • U.N. Das

    Tumoricidal action of cis-unsaturated fatty acids and its relationship to free radicals and lipid peroxidation

    Cancer Lett

    (1991)
  • Y. Suresh et al.

    Oxidant stress, antioxidants and nitric oxide in traffic police of Hyderabad, India

    Environ Pollut

    (2000)
  • Y. Suresh et al.

    Protective action of arachidonic acid against alloxan-induced cytotoxicity and diabetes mellitus

    Prostaglandins Leukot Essen Fatty Acids

    (2001)
  • I.K. Mohan et al.

    Effect of L-arginine-nitric oxide system on chemical induced diabetes mellitus

    Free Rad Biol Med

    (1998)
  • N. Madhavi et al.

    Effect of n-6 and n-3 fatty acids on the survival of vincristine sensitive and resistant human cervical carcinoma cells in vitro

    Cancer Lett

    (1994)
  • A. Khalil et al.

    Polycyclic aromatic hydrocarbons potentiate high-fat diet effects on intestinal inflammation

    Toxicol Lett

    (2010)
  • M.M. Kreusser et al.

    Differential expression of cardiac neurotrophic factors and sympathetic nerve ending abnormalities within the failing heart

    J Mol Cell Cardiol

    (2008)
  • M. Yamanaka et al.

    Protective effect of brain-derived neurotrophic factor on pancreatic islets in obese diabetic mice

    Metabolism

    (2006)
  • H.S. Oliff et al.

    Exercise-induced regulation of brain-derived neurotrophic factor (BDNF) transcripts in the rat hippocampus

    Brain Res Mol Brain Res

    (1998)
  • A. Kuroda et al.

    Brain-derived neurotrophic factor ameliorates hepatic insulin resistance in Zucker fatty rats

    Metabolism

    (2003)
  • C.L. Roth et al.

    Brain-derived neurotrophic factor and its relation to leptin in obese children before and after weight loss

    Metabolism

    (2013)
  • M. Ono et al.

    Intermittent administration of brain derived neurotrophic factor ameliorates glucose metabolism in obese diabetic mice

    Metabolism

    (2000)
  • A. Danielsson et al.

    Short-term overeating induces insulin resistance in fat cells in lean human subjects

    Mol Med

    (2009)
  • B. Kowall et al.

    Association of passive and active smoking with incident type 2 diabetes mellitus in the elderly population: the KORA S4/F4 cohort study

    Eur J Epidemiol

    (2010)
  • L. Zhang et al.

    Association between passive and active smoking and incident type 2 diabetes in women

    Diabetes Care

    (2011)
  • C. Liu et al.

    Epidemiological and experimental links between air pollution and type 2 diabetes

    Toxicol Pathol

    (2013)
  • S.A. Meo et al.

    Effect of environmental air pollution on type 2 diabetes mellitus

    Eur Rev Med Pharmacol Sci

    (2015)
  • C. Meisinger et al.

    Association of cigarette smoking and tar and nicotine intake with development of type 2 diabetes mellitus in men and women from the general population: the MONICA/KORA Augsburg Cohort Study

    Diabetologia

    (2006)
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