neonatally-Induced dIabetes : lIpId profIle outcomes and oxIdatIve stress status In adult rats

Diabetes mellitus is a metabolic disorder characterized by hyperglycemia, secretion insufficiency and receptor insensitivity to endogenous insulin1. Its incidence is associated with high morbidity and mortality rates. Increased oxidative stress is believed to play an important role in the etiology and pathogenesis of chronic complications of diabetes2,3, and is mainly characterized by imbalance between organism antioxidant defenses and oxidant molecules, known as free radicals. Free radicals are very reactive species, capable of inducing oxidation of the biological membrane of phospholipids and proteins, resulting in modifications of cell function and cellular death4,5,6. Many animal models have contributed to the elucidation of human diabetic syndromes and associated genetic factors. Among these animal models, there are those that reproduce human diabetes using streptozotocin (STZ). Probably, STZ induces diabetes by generating reactive oxygen radicals (ROS), which leads to islet cell destruction in experimental animals7. Models for rats presenting severe diabetes (fasting glycemic level higher than 200/360 mg/dL), which reproduce uncontrolled type-1 diabetes (DM1) in humans, are well established *Correspondence: Departamento de Ginecologia e Obstetrícia, FMB – Unesp Distrito de Rubião Júnior, s/n Botucatu Estado de São Paulo, Brazil CEP: 18618-000 Tel: 55143811-6181 Fax: 551438116090 damasceno@fmb.unesp.br neonatally-Induced dIabetes: lIpId profIle outcomes and oxIdatIve stress status In adult rats


IntroductIon
Diabetes mellitus is a metabolic disorder characterized by hyperglycemia, secretion insufficiency and receptor insensitivity to endogenous insulin 1 .Its incidence is associated with high morbidity and mortality rates.Increased oxidative stress is believed to play an important role in the etiology and pathogenesis of chronic complications of diabetes 2,3 , and is mainly characterized by imbalance between organism antioxidant defenses and oxidant molecules, known as free radicals.Free radicals are very reactive species, capable of inducing oxidation of the biological membrane of phospholipids and proteins, resulting in modifications of cell function and cellular death 4,5,6 .
Many animal models have contributed to the elucidation of human diabetic syndromes and associated genetic factors.Among these animal models, there are those that reproduce human diabetes using streptozotocin (STZ).Probably, STZ induces diabetes by generating reactive oxygen radicals (ROS), which leads to islet cell destruction in experimental animals 7 .Models for rats presenting severe diabetes (fasting glycemic level higher than 200/360 mg/dL), which reproduce uncontrolled type-1 diabetes (DM1) in humans, are well established utilizing high doses of STZ during the adult life of animals 8,9,10,11 .Experimental mild diabetes (fasting glycemic level from 120 to 360 mg/dL) characterizes clinical status of type 2 Diabetes mellitus (DM2).Portha et al. 12 were the first to describe an animal model for mild diabetes using neonatal STZ.Some authors administered STZ at day of birth (n0-STZ) 13,14 , 2 days after birth (n2-STZ) 15,16 , after 5 days of life (n5-STZ) 16,17 , and on the 2 nd and 9 th days of life 18 .These studies showed that, at 8 weeks and thereafter the animals presented impaired glucose tolerance and a 50% decrease in pancreatic insulin content with mild hypoinsulinemia.As reviewed by Portha et al, incompetence of the regenerated beta cells may be due to a reduced GLUT2 content, limiting glucose entry and metabolism and a decreased glucokinase affinity to glucose 19 .Experimental findings lead to the development of treatment strategies to maintain metabolic conditions as close as possible to normal.There are several reports about the use of streptozotocin to induce mild diabetes reproducing type 2 Diabetes mellitus.However, these investigations did not disclose any relation among glucose tolerance, insulin content and insulin resistance with lipid profile and oxidative stress status in adult life of rats.Therefore, this study aimed to analyze the model of mild diabetes induction in Wistar rats and to evaluate these parameters in the adult life of rats treated neonatally with streptozotocin.

metHods
Wistar male and female rats weighing about 180g (90 days of life) were adapted in our laboratory for seven days.The rats were kept in collective cages, in controlled conditions of temperature of (22 ± 3 º C), light (12 h light/dark cycles) and relative humidity (60 ± 5%).The animals were fed with laboratory chow (Purina ® ) tap water ad libitum and cared for in accordance with the principles of the Guide for Care and Use of Experimental Animals.The local Committee of Ethics in Animal Experimentation approved all experimental procedures of this study.
Female rats were mated overnight with normal male rats (parental generation).Sperm presence in vaginal wet smears in the following morning was considered as day zero (0) of pregnancy.Pregnant rats were kept in individual cages during the pregnancy period (21 days), including vaginal delivery and lactation periods (21 days).The female newborn (NB) received streptozotocin (STZ, 70 mg/kg body weight, intraperitoneally) dissolved in citrate buffer (0.1M, pH 4.5) at day 5 of life, as previously described by Murali & Goyal 17 .The terminology n5-STZ is used here to refer to this version of a model that characterizes clinical status of DM2.In the control group, NB received only citrate buffer, at day 5 of life.
In the month 3 and 4 of life, blood samples were obtained from a cut tail tip for non-fasting glycemic determinations (glucose oxidase) using a glucosimeter (One Touch Ultra -Johnson & Johnson ® ) in the morning.At the end of 4 th month of life, the glycemia were obtained immediately before anesthesia.All animals were anesthetized with sodium pentobarbital and killed.Blood samples were collected from each animal and processed for lipid profile and oxidative stress measurements.The chemicals were purchased from Wiener (Rosario, Argentina) and Sigma Chemical (St.Louis, MO, USA).Blood samples were collected in anticoagulant-free test tubes and kept at low temperature for 30 min and then centrifuged at 3,500 rpm for 10 min at 4ºC.The supernatant was collected as serum and stored at -80ºC for lipid determination.Another blood fraction was placed in anticoagulant tubes and centrifuged at 1,200 rpm for 10 min at room temperature for assay of oxidative stress biomarkers, which were estimated in the washed erythrocytes 20 .
Serum concentrations of total cholesterol, triglycerides and high-density lipoprotein cholesterol (HDL-C) were determined by the enzymatic method 21 .The absorbance of these parameters was measured at 505 nm.
Oxidative stress biomarkers evaluated were superoxide dismutase (SOD), glutathione total (GSHt) and thiobarbituric acid reactive substances as the lipid peroxidation index (TBARS).For TBARS concentration, the absorbance was measured at a wavelength of 535 nm and results were expressed as nM of thiobarbituric acid reactive species (TBARS) per gram of hemoglobin (nM/g Hb).The SOD enzymatic activity unit was defined as SOD units able to produce 50% of pyrogallol oxidation inhibition.All data were expressed in units of SOD per milligram of hemoglobin.GSHt, which consists of reduced and oxidized glutathiones, was enzymatically determined using 5.5'-dithio-bis (2-nitrobenzoic acid) (DTNB) and glutathione reductase in the presence of a reduced form of nicotinamide adenine dinucleotide phosphate (NADPH), forming 2-nitro-5-thiobenzoic acid.GSHt activity was measured at 412 nm on a spectrophotometer.One unit of its activity was equal to the micromolar of substrate reduced per gram of hemoglobin 20 .
Data are expressed as mean ± standard error of mean (SEM).The Student t test was used to determine differences between groups 22 .The limit of statistical significance was 5% (p<0.05).

Glycemia
In the 3 rd month of life, the glycemic means of rats in the n5-STZ group were significantly higher than those of rats in the control group (p<0.05).All rats in the control group exhibited glycemia under 100 mg/dL.Among the rats in the n5-STZ group, 50.0%showed glycemia under 120 mg/dL and 50.0%from 120 to 360 mg/dL.In the 4 th month of life, glycemic means of rats in the n5-STZ group remained significantly higher in relation to the control group (p<0.05).When glycemic levels were individually analyzed, alteration was observed in the distribution of glycemic ranges of rats in the n5-STZ group, since all the animals presented glycemia between 120 and 360mg/dL.Nevertheless, in the control group, the rats maintained glycemic levels similar to those found in the 3 rd month of life.Glucose levels at month 3 and 4 of life are presented in Table 1 and the glucose range distributions are presented in Table 2.

Biochemical parameters and oxidative stress biomarkers
The biochemical parameters evaluated in the 4 th month of life are presented in Table 3.Both groups did not exhibit statistical differences (p>0.05) in serum levels of total cholesterol and triglycerides.However, n5-STZ animals showed a significant decrease in HDL-cholesterol levels as compared to the control group (p<0.05).
The evaluation of oxidative stress parameters is presented in Table 4. Streptozotocin-induced diabetic rats did not present changes in lipid peroxidation (TBARS).Alterations were not observed in SOD activity or GSH-t determination when compared to the control group (p>0.05).

dIscussIon
In this study, the control animals presented normoglycemia at months 3 and 4. Control animals presented slight glycemia above 120mg/dL at the 4 th month of life however, these animals were not considered mild diabetic because glycemia was verified in the non-fasting state and our data corroborate those of other authors 17,23 .Our results showed that STZ administration on the 5 th day of neonatal life caused onset of hyperglycemia in the 3 rd month of life in rats, whose postprandial glycemic mean was of approximately 192.4 mg/dL.This model presented glycemic results similar to those found by Murali & Goyal 17 .The individual analysis of glycemia in rats showed that 50% did not present mild diabetes at the 3 rd month.Nevertheless, after the 4 th month of life, 100% of the n5-STZ rats showed glycemic levels between 120 and 360 mg/dL, similar to the glycemic mean observed by other researchers 24,25 thus ensuring the viability of the model for mild diabetes induction.Further, Weir et al. 26 demonstrated that n5-STZ rats presented lack of significant insulin re-accumulation in the pancreas, 2 weeks after the b-cell insult.
In patients, Diabetes mellitus increases the risk of developing atherosclerosis and coronary artery disease 27,28,29 , and lipids are indicated as some of the major pathogenic biological markers in situations of metabolic dysfunction (such as in insulin resistance, associated type-2 Diabetes mellitus or not) 30 .This investigation showed that n5-STZ animals presented a nonsignificant increase in total cholesterol and triglyceride levels and decreased HDL levels.With regard to the absence of alterations in the determinations of total cholesterol and triglycerides, our findings are not in agreement with those described in literature.Insulin resistance and Diabetes mellitus affect virtually every lipid and lipoprotein and, therefore, dyslipidemia is present in most diabetic patients 27,31,32 .Dyslipidemia is reflected by elevated levels of triglycerides, VLDL and LDL-cholesterol and lower HDL-cholesterol levels 33 .Although our results do not reflect comments in clinical studies, the Brazilian Society of Cardiology acknowledges that decreased HDL (isolated or in association with increased levels of LDL and/or triglycerides) characterizes the laboratory classification of dyslipidemia 34 .HDL cholesterol and apo AI levels are characteristically reduced in insulin-resistant people.Much of this derives, as in the case of low dense LDL, from the action of CETP (cholesterol ester transferase) mediated transfer of cholesteryl ester from HDL to triglyceride rich lipoproteins (chylomicrons and VLDL).A consistent finding is the inverse relationship between plasma insulin (or C-peptide) concentrations, which are measures of insulin resistance and HDL-cholesterol levels 35 .
There is increasing evidence, in both experimental and clinical studies, to suggest that oxidative stress plays a major role in the pathogenesis of Diabetes mellitus.In this study, TBARS levels in the n5-STZ rats remained unaltered.Our result suggests that the neonatal STZ-induced diabetes model was insufficient to increase glycemia and so, exacerbate oxidative stress.However, increase of glycemia to a moderate level was verified.With respect to SOD, the enzyme responsible for neutralizing   superoxide anion levels, this study showed no changes in SOD activity in the n5-STZ rats.These results corroborate with literature, which observed no significant changes in SOD activity in diabetic rats 36,37 .However, there are also conflicting findings in literature.There is evidence of reduced SOD activity in alloxaninduced diabetic rats 38 , and higher activity was observed in DM1 and DM2 subjects 39 .The glutathione antioxidant system has a fundamental role in cellular defense against reactive free radicals and other oxidant species 40 .GSHt concentration also presented no change in the n5-STZ rats in this investigation.Damasceno et al. 6 observed reduction in GSH determination in STZ induced-severe diabetic rats.Furthermore, it has been suggested that there may be temporal changes in enzyme activity that are both transitory and biphasic in nature.For instance, after prolonged hyperglycemia in severe diabetes (glycemia>360 mg/dL), induction of certain antioxidant enzymes or a return to normal values from previously decreased values may occur as a compensatory mechanism in response to constant exposure to increased stress.This could explain the decrease, increase or normality in SOD and GSH observed by different investigators.Thus, the present study showed that the neonatal streptozotocin-induced mild diabetes model in rats caused lipid profile alteration in the HDL-cholesterol level, but it was not sufficient to impair antioxidant enzyme activity or peroxidation lipid determination sampled from adult Wistar rats.

conclusIon
The present investigation confirmed a mild diabetes status since the 4 th month of life in rats.This study contributed to understanding the relation between lipid profile and oxidative stress status in the mild/moderate diabetes model for laboratory animals as well as to a better comprehension of the pathophysiological mechanisms of mild diabetes or hyperglycemia.

Table 2 -Distribution of rats in relation to non-fasting glycemic levels. rats treated with citrate buffer solution (control group) and streptozotocin (n5-STZ group) in the neonatal period (mean ± SEm)
% of rats presenting glycemia with indicated values in the 3 rd and 4 th months of life.*p<0.05-statistically significant difference compared to month 3. a

Table 3 -Biological lipid profile in the 4 th month of life in rats treated with citrate buffer solution (control group) and streptozotocin (n5-STZ group) in the neonatal period (mean ± SEm)
*p<0.05 -statistically significant difference compared to control group.

acKnowledgements
The authors are grateful to Mariana Takaku and Marisa Akemi Takeno, graduate students at the Botucatu Medical School -Unesp; Mr. Carlos R. Gonçalves Lima, from the Experimental Laboratory of Infectious Diseases, for technical support, and to the Research Support Center (RSC) for their valuable contribution in study design and statistical analysis.