Effects of leptin on oxidative stress in healthy and Streptozotocin ...

Mol Cell Biochem (2007) 302:59–65 DOI 10.1007/s11010-007-9426-5

Effects of leptin on oxidative stress in healthy and Streptozotocin-induced diabetic rats S¸ ebnem Gu¨len Æ Sibel Dinc¸er

Received: 1 December 2006 / Accepted: 2 February 2007 / Published online: 24 February 2007 Springer Science+Business Media B.V. 2007

Abstract Aims/hypothesis It is generally accepted that oxidative stress is responsible for etiology and complications of diabetes. During uncontrolled Type 1 diabetes, plasma leptin levels rapidly fall. However, it is not known whether diabetes-induced hypoleptinemia has any role in oxidative stress related to uncontrolled Type I diabetes. The present study was designed to examine the effects of leptin treatment on plasma lipid peroxidation and reduced glutathion of normal and streptozotocin(STZ)-induced diabetic rats. Methods Diabetes was induced by single injection of Streptozotocin (55 mg/kg bw). One week after induction of diabetes, rats began 5-day treatment protocol of leptin injections of (0.1 mg/kg bw i.p.) or same volume vehicle. At the end of the 5th day, rats were sacrificed by cardiac puncture under anesthesia and their plasma was taken for plasma leptin, malondialdehyde, and reduced glutathione measurements. Results Plasma leptin levels decreased in STZ-induced diabetic rats while plasma glucose, TBARS, and GSH levels increased. Plasma leptin levels were not affected with leptin treatment in both diabetic and non-diabetic rats. The elevation in plasma TBARS associated with STZ diabetes decreased with leptin treatment. Leptin also increased plasma GSH levels in diabetic rats. In nondiabetic rats, treatment with leptin did not change S¸ . Gu¨len S. Dinc¸er Department of Physiology, Faculty of Medicine, Gazi University, Ankara, Turkey Present Address: S¸ . Gu¨len (&) Department of Physiology, Faculty of Medicine, Baskent University, 06530 Ankara, Turkey e-mail: [email protected]

plasma TBARS and GSH levels. Conclusions/interpretations In conclusion, leptin treatment is able to attenuate lipid peroxidation in STZ-diabetic rats, in the onset of diabetes, by increasing the GSH levels without affecting hyperglycemia and hypoleptinemia. Keywords Oxidative stress Diabetes mellitus Streptozotocin Leptin Plasma Malondialdehyde Reduced glutathione Rat Abbreviations STZ (Streptozotocin)

Introduction Diabetes is a chronic metabolic disorder and a major worldwide health problem. It is characterized by absolute or relative deficiencies in insulin secretion and/or insulin action associated with chronic hyperglycemia and disturbances of carbohydrate, lipid, and protein metabolisms. As a consequence of metabolic derangements in diabetes, various complications develop including both macro and micro-vascular dysfunctions [1]. Oxidative stress results from an imbalance between the generations of oxygen derived radicals and the organism’s antioxidant potential. The free radicalmediated peroxidation of membrane lipids increases membrane fluidity and permeability with loss of its integrity [2, 3]. The process of lipid peroxidation is one of the oxidative conversion of polyunsaturated fatty acids to products known as malondialdehyde (MDA). MDA is a highly toxic molecule and its secondary products such as thiobarbutiric acid reactive substance

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(TBARS), are commonly used to evaluate lipid peroxidation [4, 5]. Glutathione (GSH) is a major nonenzymatic component of the cellular antioxidant system, playing an important role in the antioxidation of ROS and free radicals [6, 7]. Various studies have shown that both insulin dependent (Type 1) and insulin non-dependent diabetes (Type 2) are associated with increased formation of free radicals and decrease in antioxidant potential [8, 9]. Tissue damage induced by oxidative stress has also been implicated in the pathogenesis of diabetic complications [1, 10, 11]. Various plasma protein and lipids were reported to be oxidatively modified both in experimental and clinical studies [12, 13]. Oxidative stress is increased in diabetes because of multiple factors. Hyperglycemia itself generates reactive oxygen species (ROS), which in turn cause lipid peroxidation and membrane damage via autooxidation of glucose [14]. Glucose auto-oxidation has been linked to non-enzymatic glycosylation and glycosylated proteins have been shown to be a source of free radicals [15–18]. Apart from increased nonenzymatic and auto-oxidative glycosylation, metabolic stress due to decreased cellular antioxidant levels and reduction in the activity of enzymes that dispose of free radicals could lead to oxidative stress in diabetes [19]. Streptozotocin (STZ)-diabetic animals, often used as a study model of Type 1 diabetes, are characterized not only by decreased insulin levels and hyperglycemia, but also by decrease in circulating leptin levels [20, 21]. Leptin is predominantly expressed by adipocytes, and its plasma levels correlate well with the body fat mass [22, 23]. It was previously suggested that this decrease in leptin contribute to hyperphagia in diabetic rats [24]. Administration of leptin results in partial correction of food intake, as well as normalization of postabsorbtive plasma glucose levels in STZ-induced diabetes [25]. However, leptin cannot only improve the metabolic profile, but also diminishes insulin requirements for reestablishing normoglycemia in STZ-diabetic mice [26]. The relationship between leptin and oxidative stress has not been clear yet. Changes in the antioxidant defense mechanisms in plasma of ob/ob mice [27] and patients [28] with human leptin gene mutation have been reported previously. The above-mentioned studies demonstrated that leptin administration corrected alterations seen with antioxidant activities. Several in vitro studies have demonstrated that leptin stimulates ROS production by inflammatory cells [29], endothelial cells [30], and other cell types [31]. There are no studies on the role of leptin on oxidative stress in STZ-induced diabetes. The aim of our

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Mol Cell Biochem (2007) 302:59–65

present study was to investigate the role of exogenous mouse recombinant leptin on plasma TBARS and GSH levels of non-diabetic and STZ-induced diabetic rats.

Materials and methods Animals Prior approval for this experiment was obtained from the Animal Experimentation Ethics Committee at Gazi University. Adult male Wistar albino rats (age, 8–9 weeks; weight, 250 ± 6.5 g, mean ± SEM) were obtained from Refik Saydam Hıfzıssıhha Institute (Ankara/Turkey). Rats were housed individually in plastic cages with 12-h/12-h light–dark cycle (light from 8.00 a.m. to 8.00 p.m.), with room temperature at 22 ± 2C. Rats were allowed to acclimatize for 1 week and were offered food and water ad libitum throughout the study. Chemicals Streptozotocin was procured from Sigma Chemical Co., St. Louis, MO., USA. STZ dissolved in a solution of cold citrate buffer (0.1 M, pH = 4.5). These solutions were freshly dissolved and shielded from light. Mouse recombinant leptin was purchased from Calbiochem (Germany), Reconstitution of leptin was carried out by adding 15 mM sterile HCl (0.5 ml/ 1 mg). After dissolving the protein, 7.5 mM sterile NaOH (0.3 ml/1 mg) was added. The dissolved protein was then stored at –20C. The hormone was diluted in phosphate-buffered saline (PBS) (pH: 7.4) as 250 lg in 1 ml just before use. All the other reagents were obtained from Sigma Chemical Co., St. Louis, MO., USA. Diabetes procedure Animals were divided into two main groups as nondiabetic control (NDM) (n = 12) and STZ-induced diabetic (STZ-DM) (n = 12) rats. After overnight fasting (deprived of food for 18 h had been allowed free access to water), diabetes was induced by intraperitoneally (i.p.) injection of STZ at a dose of 55 mg/ kg body weight. Control rats were injected with citrate buffer alone. On the 7th day after i.p. administration of STZ, the plasma concentration of glucose was measured using a Glucometer (Glucotrend brand). Rats with blood glucose levels of 300 mg/dl or higher were considered diabetic.


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Treatment procedure

Statistical analysis

Rats were divided into four groups: 1—non-diabetic control rats treated with vehicle (phosphate-buffered saline) (NDM-VEH; n = 6), 2—non-diabetic rats treated with recombinant murine leptin (NDM-LEP; n = 6), 3—STZ-induced diabetic control rats treated with vehicle (STZ-VEH: n = 6), 4—STZ-induced diabetic rats treated with leptin (STZ-LEP; n = 6). The treatment started on the 7th day after STZ-injection and this was considered as the 1st day of treatment. Six of the control and six of the diabetic group were treated with leptin (0.1 mg/kg bw/day), and the remaining animals in each group received vehicle at the same volume only. Leptin and the vehicle were administered between 3.00 and 3.30 p.m. for 5 days. At the end of the 5th day, STZ-treated animals and the corresponding controls were all handled in the same way and they were sacrificed by cardiac puncture under thiopental anesthesia (50 mg/kg bw).

All data are expressed as group means ± SEM. Statistical analyses were performed using the statistical package for the social science software (SPSS) program version 14.0. The results were evaluated using Independent t-test, Mann–Whitney U-test, and ANOVA variance analysis. Statistical significance was considered at P < 0.05.

Results Blood glucose levels Diabetes was confirmed by the presence of hyperglycemia after 1 week of injection of STZ. Blood glucose level was significantly higher (P < 0.0001) in diabetic rats than in controls (Fig. 1). Leptin treatment did not change the blood glucose levels in NDM and STZ-DM groups.

The measurement of plasma leptin Plasma leptin levels Plasma leptin levels were measured using a rat leptin ELISA kit (Titerzyme, Assay Designs, USA.) The measurement of plasma TBARS The method described by Kurtel et al. was used. One milliliter of thiobarbutiric acid/tricloracetic acid/ hydrochloric acid was added to 500 ll plasma in a microcentrifugation tube and then vortexed for 30 s. The mixture was centrifuged at 10,000 rpm for 5 min and the supernatant was placed into a glass tube. Ten micro liters of butylated hydroxytoluene was added to this mixture and boiled for 15 min. The sample was evaluated in comparison with distilled water at 532 nm in an Elisa reader, and the MDA level was calculated using the following formula: Plasma MDA = adsorbance X 19.2 nmol MDA/ml plasma [32].

Diabetic control rats showed a significant decrease in plasma leptin levels when compared to the non-diabetic control groups (P < 0.001). Leptin treatment did not show any effect on plasma leptin levels in both non-diabetic and diabetic rats. Plasma MDA and GSH levels Lipid peroxidation, as reflected by plasma TBARS (P < 0.001) and cellular antioxidant GSH (P < 0.01) values was higher in diabetic rats than that of their healthy controls. Leptin treatment did not show any statistically significant effect on TBARS, and GSH

*

The measurement of plasma GSH The method described by Kurtel et al. was used. Plasma (500 ll) and 1 ml hydrochloric acid/Na dodesil sulfate/EDTA (pH = 8.2) were placed in a microcentrifugation tube and centrifuged at 25C and 12,000 rpm for 5 min. Ditionitrobenzoic acid (0.3 mM) was added into the supernatant and the glass tube was kept at 37C for 20 min. The sample was read at 405 nm in an ELISA reader, and the GSH levels were calculated using the following formula: Plasma glutathione = 3.18 · adsorbance/0.0136 nmol/ml [32].

Blood Glucose (mg /dl)

450 400

NDM

STZ-DM

350 300 250 200 150 100 50 0 NDM

STZ-DM

Fig. 1 Changes in the blood glucose levels in non-diabetic (NDM; n = 12) and diabetic (STZ-DM; n = 12) rats. Results are expressed as mean ± SEM. * P < 0.0001 NDM vs STZ-DM

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levels in non-diabetic rats while it caused significant decrease in plasma TBARS levels (P < 0.01), significant increase in plasma GSH levels (P < 0.01). Table 1 shows the levels of leptin, TBARS and reduced GSH in plasma and blood glucose of control and experimental groups of rats.

Discussion Streptozotocin is a valuable agent for experimental induction of Type I diabetes. STZ treatment destroys the beta cells of the pancreas and STZ-induced diabetic rats are considered a model of Type I diabetes [33]. Increase in blood glucose is a typical feature in diabetes and, in our study as anticipated, STZ administered rats showed 5-fold increases approximately in blood glucose by the end of the first week. Plasma leptin levels also decrease in Type I diabetes like insulin levels and body fat content [20, 21]. Moreover, this decrease in leptin contributes to hyperphagia in diabetic rats [24]. We also observed a significant decrease in plasma leptin levels of streptozotocin-induced diabetic rats. Streptozotocin-induced hypoleptinemia may be related to a reduced adipose tissue mass, reduced assimilation, and storage of energy substrates in the far tissue in insulin deficiency and/or direct toxic effect of streptozotocin on the adipose tissue [20, 21, 34]. In diabetes, protein glycation and glucose autooxidation may induce free radical formation leading to lipid peroxidation [3, 12]. In our experimental model, induction of diabetes resulted in apparent oxidative conditions. We found an increase in TBARS levels as an index of lipid peroxidation in plasma of STZ-diabetic rats. It has been reported that tissue and plasma MDA levels of STZ-induced diabetic rats are increased by lipid peroxidation [2, 35]. Increased levels of TBARS—circulating products of lipid peroxidation—suggests that increased oxidative stress in experimental diabetic animals. These observations agree with large number of studies

finding increased plasma TBARS or MDA in diabetic patients [36, 37]. Further, we also measured the GSH levels in plasma of rats, since the glutathione plays an important role in cellular defense against reactive free radicals and other oxidant species [38, 39]. In our study, plasma GSH concentration of STZ-induced diabetic rats is significantly higher than that of non-diabetic rats. Since marked alterations in antioxidant enzyme activities and tissue GSH concentrations have been reported in diabetes, tissue antioxidant systems emerges as an important factor in the etiology of diabetic complications [38–41]. In our study, increased GSH in plasma of diabetic rats could be the result of adaptive mechanism in response to oxidative stress. It was demonstrated that intracellular GSH levels increased and free radical formation decreased via increase in glutamate sistein ligase enzyme activity in cells exposed to oxidative stress by prooxidants [42, 43]. The contribution of hypoleptinemia developed in uncontrolled Type-1 diabetes, which is a hypoinsulinemic and hyperglycemic condition, on oxidative stress is not clear. Nonetheless, not enough studies have been done concerning the relationship between oxidative stress and leptin administration in diabetes. According to our study, decreased leptin levels observed in STZinduced diabetes may be related with increased lipid peroxidation and compensatory increased GSH levels. In the present study, diabetic rats were treated with a mouse recombinant leptin for 5 days, which result in a significant but incomplete reduction of oxidative stress parameters in plasma. Restoration of the defective antioxidant enzyme activity after leptin treatment in plasma of ob/ob mice and humans with leptin gene mutation has been reported previously [27, 28]. Ozata et al. suggested that leptin modulates the activity of several antioxidant enzymes such as plasma and erythrocyte glutathione peroxidase (GPx) and erythrocyte copper/zinc superoxide dismutase (SOD) in patients with leptin gene mutation [28]. These changes are very likely the result of leptin’s modulatory effect on metabolic and

Table 1 Mean levels of blood glucose, plasma leptin, TBARS, and GSH for all groupsa Groups

n

Blood glucose (mg/dl)

Plasma leptin (ngr/dl)

Plasma TBARS (nmol/ml)

Plasma GSH (nmol/ml)

NDM-VEH NDM-LEP STZ-VEH STZ-LEP

6 6 6 6

123.16 ± 3.05 129.66 ± 8.96 341.83 ± 33.25** 357.5 ± 19.49

3.34 ± 0.43 2.47 ± 0.68 0.34 ± 0.11** 0.2 ± 0.05

0.36 0.34 0.80 0.45

150.5 172.2 174.4 242.4

a

Mean ± SEM STZ-VEH vs. NDM-VEH; * P < 0.01, ** P < 0.001 STZ-LEP vs. STZ-VEH; *** P < 0.01

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± ± ± ±

0.01 0.02 0.1** 0.03***

± ± ± ±

7.41 11.22 4.44* 22.97***


hormonal disturbances in case of leptin gene mutation. It has been shown that administration of recombinant leptin reduces food intake leading to weight loss and has a reducing effect on oxidative stress in high fat diet induced oxidative stress [44]. Leptin synthesized and secreted mainly by adipose tissue in proportion to fat stores. Hyperleptinemia is a prominent feature of obesity and it is likely to be involved in the pathogenesis of obesity-related pathologies [22]. Studies like that of Beltowski et al., in which hyperleptinemia is induced by the administration exogenous leptin in nonobese animals, suggest that leptin increases the level of systemic oxidative stress [45, 46]. Furthermore, several in vitro studies have demonstrated that in the presence of high leptin concentration, ROS production was stimulated by inflammatory cells, endothelial cells, and other cell types [29–31]. Above-mentioned studies pointed out that the relationship between leptin and oxidative stress is associated with circulating or media leptin concentration and metabolic profile. Interestingly, TBARS and GSH levels were not significantly altered with leptin administration in normoglycemic and normoleptinemic conditions in our experimental protocol. Treatment with leptin had no effect on plasma leptin and blood glucose levels neither in non-diabetic nor diabetic rats in this experiment. Chinookoswong et al. demonstrated that leptin administration can normalize blood glucose levels when just pharmacologic doses [25] used in STZ-diabetic rats while we used leptin in physiologic dose [47]. In addition, different from that study, we administered leptin by a single intraperitoneal injection for 5 days, but they applied it by the continuous infusion. It is possible that in our protocol, the leptin concentration probably increased markedly soon after injection, but later declined in plasma, favored its effect on oxidative stress. Our findings showed for the first time that, administration of leptin can markedly reduce the STZdiabetes-induced oxidative stress in rats without altered plasma leptin and glucose levels. The present study supports the hypothesis that oxidative stress exists in STZ-diabetic rats due to the elevation of MDA and GSH but, interestingly, although blood glucose levels were similar in the two groups of diabetic rats. Lipid peroxidation levels were significantly lower and GSH levels were significantly higher in the group receiving leptin, indicating that the inhibitory effect of leptin on plasma lipid peroxidation in diabetic rats could result, in part, from elevation of the GSH content. The present study has several important factors which need to be mentioned. First, we have used the

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murine leptin, and the hormone originating from different species could have different actions. In fact, we did not know whether recombinant leptin may be active as much as native hormone. Second, leptin was administered via i.p. bolus injection which could have been resulted in significant variations of its plasma concentration. IP-injected leptin is expected to be transported to the liver, where it can, at least theoretically, influence other oxidative and antioxidative parameters in different glycemic conditions. Third, although we demonstrated several effects in leptintreated animals, the treatment dose applied in the study was reported to be in the physiological ranges. It should not be ignored; decreases of leptin and related metabolic disturbances in STZ-diabetic animals require insulin administration in proportion to the degree of glucose lowering [20]. Fifth, we performed this study on lean and short-term diabetic rats and we administered leptin only for 5 days. The long-term effects of STZ were not evaluated. Since diabetes is a chronic illness and complications occur in the long term, it is necessary for the long-term use of leptin in diabetics to be investigated. Finally, as mentioned above, the results of diabetic studies are extremely variable, depending on experimental conditions. In conclusion, we observed that uncontrolled diabetes of 2 weeks duration in rats is associated with enhanced lipid peroxidation, hyperglycemia, and hypoleptinemia. Our experimental data suggest that the mouse recombinant leptin attenuates diabetes induced lipid peroxidation by increasing the levels GSH without affecting hyperglycemia and hypoleptinemia in these rats. The results presented here suggest the possible paradoxical effects of leptin in different glycemic and/or oxidative stress conditions. However, it is not clear whether these effects are due to direct effects of leptin or secondary to interactions with other physiological factors. Finally, there is a need for further research on long-term use and on the mechanism(s) of the antioxidant effects. Acknowledgment This study was supported by Gazi University Research Foundation. (GU-BAP 01/2002-58)

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