Curcumin prevents streptozotocin-induced islet damage by scavenging free radicals: A prophylactic and protective role

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Abstract

Pancreatic islet cell death is the cause of deficient insulin production in diabetes mellitus. Approaches towards prevention of cell death are of prophylactic importance in control and management of hyperglycemia. Generation of oxidative stress is implicated in streptozotocin, a beta cell specific toxin-induced islet cell death. In this context, antioxidants raise an interest for therapeutic purposes. Curcumin, a common dietary spice is a well known antioxidant and hence we investigated its effect on streptozotocin-induced islet damage in vitro. Isolated islets from C57/BL6J mice were incubated with curcumin for 24 h and later exposed to streptozotocin for 8 h. The effect of streptozotocin exposure to islets was determined with respect to islet viability and functionality, cellular reactive oxygen species concentrations and levels of activated poly (ADP-ribose) polymerase-1. Cellular antioxidant potential (Cu/Zn superoxide dismutase) and advanced glycation end-product related damage was assessed to determine the metabolic status of treated and untreated islets. Islet viability and secreted insulin in curcumin pretreated islets were significantly higher than islets exposed to streptozotocin alone. Curcumin retarded generation of islet reactive oxygen species along with inhibition of Poly ADP-ribose polymerase-1 activation. Although curcumin did not cause overexpression of Cu/Zn superoxide dismutase, it prevented reduction in levels of cellular free radical scavenging enzymes. Our data shows that curcumin protects islets against streptozotocin-induced oxidative stress by scavenging free radicals. We show here for the first time, that prophylactic use of curcumin may effectively rescue islets from damage without affecting the normal function of these cellular structures.

Introduction

Type 1 diabetes mellitus (T1D) is an autoimmune disorder caused by lymphocytic infiltration and beta cell destruction of the pancreatic islets of Langerhans. This beta cell destruction in vitro may be effected through exposure to pro-inflammatory cytokines such as TNF-alpha and IL-1beta (Herold et al., 1996) or by extensive generation of reactive oxygen species (Eizirik and Mandroup-Poulsen, 2001). It is known that pancreatic beta cells contain very low levels of antioxidant enzymes which render them more susceptible to reactive oxygen species-induced toxicity as compared to other cell types (Tiedge et al., 1997). Several studies have shown that streptozotocin causes increased generation of reactive oxygen species, DNA damage and consequently extensive DNA repair leading to depletion of intracellular nicotinamide adenine dinucleotide (NAD) pool which may mediate streptozotocin-induced toxicity. This depletion in NAD level causes severe depletion of the ATP pool, activating the DNA repair enzyme Poly ADP-ribose polymerase-1. Overactivation of Poly ADP-ribose polymerase-1 has been implicated in non-obese diabetic (NOD) (Suarez-Pinzon et al., 2003) and multiple low dose streptozotocin-induced diabetes, the inhibition of which results in prevention of diabetogenesis (Mabely et al., 2001). Majority of the beta cell damage is attributed to the extensive oxidative stress caused by streptozotocin-induced depletion of free radical scavenging potential and Poly ADP-ribose polymerase-1 overactivation. Since Poly ADP-ribose polymerase-1 inhibitors are known to prevent such streptozotocin-induced damage (Uchigata et al., 1982), utilization of antioxidants for rescue of beta cells arises as an interesting possibility. Antioxidants, including vitamins C and E have been used with some success for reduction of oxidative stress and protection of beta cells against streptozotocin insults in vivo (Kaneto et al., 1999, Cay et al., 2001, Kinalski et al., 1999). Other antioxidants including N-acetyl-l-cysteine are known to protect against nitrosative stress and associated secondary complications in experimental diabetes (Nagareddy et al., 2006). Overexpression of antiapoptotic or antioxidant proteins such as thioredoxin (Chen et al., 2005), catalase (Chen et al., 2001), and metallothionine (Kasano et al., 2004) rescues beta cells from streptozotocin insult in vivo. Additionally, treatment with antioxidants also salvages islets from streptozotocin insult in vitro. For example Nicorandil, an antianginal and antioxidant agent partially rescued rat islet beta cells from streptozotocin-induced damage in vitro by virtue of its radical scavenging effect (Kasano et al., 2004). Melatonin and polyenoylphosphatidylcholine (PPC) counteract streptozotocin-induced inhibition of glucose metabolism and insulin release in cultured rat islets (Andersson and Sandler, 2001) and cultured Min6 cells (Lee et al., 2003) etc.

Curcumin (diferuloylmethane) is a naturally occurring yellow pigment isolated from the rhizomes of the plant Curcuma longa (Linn) found in south Asia (Lodha and Baggha, 2000) and is a potent antioxidant agent and free radical scavenger (Fujisawa et al., 2004). Along with being an inhibitor of lipid peroxidation (Sreejayan and Rao, 1994), it is also an inhibitor of nitric oxide synthase (NOS) overexpression (Pan et al., 2000) and of nuclear factor kappa B activation (Weber et al, 2006). The efficacy of curcumin has been widely observed in reducing various diabetic secondary complications such as diabetic nephropathy/renal lesions (Sharma et al., 2006), retinopathy (Kowluru and Kanwar, 2007), wound healing (Panchatcharam et al., 2006), and reduction of advanced glycation end products (Sajithal et al., 1998). Its potential as a hypoglycemic agent has also been studied in animals (Pari and Murugan, 2005, Hussain, 2002) and humans (Srinivasan, 1972) with conflicting results. Furthermore, although many aspects of curcumin-induced cytoprotection are studied, its efficacy in protecting isolated islets against streptozotocin-induced free radical-mediated damage has not been demonstrated so far. We hypothesized that curcumin may protect isolated islets against streptozotocin-induced oxidative stress and resulting beta cell damage and dysfunction. Logically, curcumin would impart some protection against oxidative damage to beta cells, but it would be of interest to investigate the mode of curcumin-induced cytoprotection. In this study, we have investigated the protective action of curcumin on streptozotocin-induced islet cell damage in vitro. We show here for the first time that curcumin treatment for 24 h prior to streptozotocin exposure rescues cultured mouse islets from damage and dysfunction by virtue of its free radical scavenging activity.

Section snippets

Animals

Six to eight week old C57/BL6J male mice were obtained from an inbred colony at the National Centre for Cell Science, India and given free access to standard feed and water. The care and use of all experimental animals complied with relevant animal welfare laws. All animals were maintained in a pathogen-free environment on a 12 h light:12 h dark cycle.

Reagents

Streptozotocin, collagenase, curcumin, DCFH-DA etc. were purchased from Sigma-Aldrich Corp., St Luis. MO, USA. RPMI 1640 with HEPES, glucose and

Curcumin prevents streptozotocin-induced islet damage by scavenging cellular reactive oxygen species

We treated islets with increasing doses of curcumin for various time points and estimated the effect of curcumin pretreatment on percent viability, corresponding cellular reactive oxygen species and insulin secretion in islets exposed to 1 mM streptozotocin for 8 h. Streptozotocin exposure led to about 50% reduction in viability as compared to control untreated islets, while pretreatment with curcumin rescued islets from streptozotocin-induced death in a dose dependant manner (P < 0.001). Maximum

Discussion

Although many reports confirm the efficacy of curcumin in cytoprotection of various cell lines against toxicity (Grandjean-Laquerriere et al., 2002, Yadav et al., 2005, Masamune et al., 2006) few studies report the efficacy of curcumin in mitigating streptozotocin-induced damage to isolated pancreatic islets. Most diabetes related studies using curcumin show the efficacy of oral administration at various doses for reduction of secondary complications in streptozotocin-induced diabetic animals (

Acknowledgment

Authors wish to acknowledge Dr. G.C. Mishra, for use of institutional facilities. We also acknowledge Dr. A. Hardikar for valuable suggestions and correction of manuscript.

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