Poster # T2027

Comparison of the effects of acute and chronic treatments with taurine and nicotinamide on brain lipid peroxidation and antioxidant status of diabetic rats

Sanket N. Patel, Kashyap Pandya and Cesar A. Lau-Cam Department of Pharmaceutical Sciences, St. John's University, College of Pharmacy and Health Sciences, Jamaica, New York, NY 11439

Introduction ♦ Nicotinamide (NIC) (I) is a form of vitamin B3 that has been extensively

investigated in humans and animal models of diabetes for the ability to prevent or delay type 1 (insulin-dependent) diabetes and to protect islet cells against cytotoxicity in vitro (Kolb and Burkart, 1999).

♦ NIC has been successfully used to inhibit or mitigate the selective pancreatotoxicity of alloxan and streptozotocin, two compounds commonly used in the laboratory for the chemical induction of diabetes (Lenzen, 2008); and in large doses it was reported to suppress the development of diabetes, to reverse overt diabetes when diagnosed at an early stage, and to reduce insulitis in NOD mice (Yamada et al., 1982). However, some laboratories have found that NIC delays rather than completely prevents the development of the disease or reverses it (Kolb and Burkart, 1999).

♦ There is also evidence to suggest that in micromolar concentrations NIC can inhibit oxidative damage of rat brain mitochondrial membrane proteins and lipids by reactive oxygen species (ROS) generated in vitro by ascorbate-Fe(II) and photosensitizing systems, an effect that was greater than that provided by endogenous antioxidants such as ascorbate and α-tocopherol (Kamat and Devasagayam, 1999). Moreover, using an experimental model of diabetic peripheral neuropathy, a 2-weeks treatment with NIC (200-400 mg/kg/day) was shown to correct the increase in sciatic nerve lipid peroxidation (LPO,) assessed by measuring malondialdehyde and 4-hydroxyalkenal levels, along with nerve perfusion deficit and nerve conduction slowing

(Stevens et al., 2007).

♦ Taurine (TAU) (II) is an endogenous nonprotein amino acid which has demonstrated a multitude of biological actions of benefit in diabetes. Experimental work in animal models of spontaneous and chemically-induced diabetes have indicated that in addition to attenuating glucose- and insulin-related metabolic abnormalities, LPO and the formation of advanced glycation end products, TAU also demonstrates antioxidant and membrane stabilizing properties, the ability to stimulate islet cell proliferation, to reduce pancreatic cell apoptosis and to reduce insulitis when consumed as a diet supplement early in life (Pandya et al., 2000; Shaffer et al., 2009; Sirdah, 2104). In view of high susceptibility of the CNS to oxidative stress and of the particular abundance of TAU in the brain and spinal cord, it has been suggested that TAU may serve as a central intra-cellular antioxidant (Biasetti and Dawson Jr., 2002). In this context, TAU has been found to ameliorate the LPO that develops in several brain areas as the result of the inflammatory response elicited by the endotoxin lipopolysaccharide in rats (Cetin et al., 2012).

I II

Objectives Taking into account the antioxidant actions that NIC and TAU have demonstrated in vitro and in vivo, the present study was under-taken in type 2 diabetic rats to:

[1] Determine the pattern and extent of oxidative stress in the spinal cord and different areas of the brain;

[2] Ascertain the actions of NIC and TAU in protecting the CNS against diabetes-induced oxidative stress;

[3] Compare the protective actions of NIC and TAU in the diabetic CNS following short-term (acute) and long term (chronic) chronic administration;

[4] Verify the extent to which the actions of NIC and TAU in the diabetic CNS differ according to the area of the CNS examined.

Experimental ♦ Animals – Male Sprague-Dawley rats, 225-250 g, acclimated for 1 week

in a room maintained at a constant humidity and temperature. The rats had free access to a commercial rodent diet (LabDiet® 5001, PMI Nutrition International, Brentwood, MO) and filtered tap water. For experimental purposes they were randomly assigned to groups of 6 rats each.

♦ Treatment agents – Streptozotocin (STZ) in 10 mM citrate buffer pH 4.5, NIC and TAU in physiological saline.

♦ Treatment groups, doses and schedules – In acute studies, rats in the treatment group received a single intraperitoneal (i.p.) dose of either NIC (250 mg/kg) or TAU (2.4 mM/kg) followed 45 min later by an i.p. dose of STZ (60 mg/kg). Rats assigned to a diabetic group received only 2 mL of physiological saline by the i.p. route prior to STZ. Rats in the control group received only 2 mL of physiological saline by the i.p. route. At 24 hr after a treatment with STZ the rats were anesthetized with isoflurane and sacrificed by decapitation.

♦ In chronic studies, groups of 6 rats each received a single i.p, 60 mg/kg, dose of STZ on day 1. Starting on day 15 and continuing until day 56, the diabetic rats received either an oral daily dose of NIC (250 mg/kg) or of TAU (2.4 mM/kg). Rats in the control group received 2 mL of 10 mM citrate buffer pH 4.5 by the i.p. route on day 1. All the rats were sacrificed by decapitation under isoflurane anesthesia 24 hr after the last treatment.

♦ Samples – Blood samples were collected in test tubes containing disodium EDTA and processed for their plasma fractions, which was used for the assay of glucose. Brains and spinal cords were surgically excised after cracking the skulls open. Each brain was divided into cerebellum, cortex, and stem. All the tissues were frozen in liquid nitrogen and stored in a deep freezer at -50°C until needed. Each part was made into a 1: 30 (w/v) homogenate in 0.01 M PBS pH 7.4 containing 0.05 M EDTA disodium with an electric homogenizer, the suspension was centrifuged at 3000 x g and 4oC for 10 min, and the supernatant was used to measure nonenzymatic (i.e., MDA, NO, GSH, GSSG) and enzymatic (i.e., catalase (CAT), superoxide dismutase (SOD),

glutathione peroxidase (GPx)) indices of oxidative stress.

Assays (a) Plasma glucose – It was measured using a commercial colorimetric

assay kit (Procedure No. 510 from Sigma-Aldrich, St. Louis, MO), which is based on the method of Raabo and Terkildsen (1960).

(b) Tissue MDA – It was measured as thiobarbituric acid reactive substances (TBARS) by the endpoint assay method of Buege and Aust (1978).

(c) Tissue NO – It was measured indirectly as nitrite using the Griess reagent and the experimental conditions given by Fox et al. (1981).

(d) Tissue GSH – It was measured by the fluorometric method of Akerboom and Sies (1981) after its reaction with o-phthaldehyde. GSSG was measured in a separate aliquot of the same sample, after removal of any preexisting GSH by a reaction with N-ethylmaleimide as described by Guntherberg and Rost 1966).

(e) Tissue CAT activity – It was measured by the method of Aebi (1984), which is based on the rate of disappearance of exogenous H2O2 added to the sample;

(f) Tissue GPx activity – It was measured according to the method of Günzler and Flohé (1985), based on the reduction of GSSG to GSH in the presence of NADPH;

(g) Tissue CuZn-SOD activity – It was measured by the spectrophoto-metric method of Misra (1985), which measures the ability of SOD to prevent

the autoxidation of epinephrine by superoxide anion radical to a pink adrenochrome showing a strong absorbance at 480 nm.

Statistical analysis of the results Results are reported as mean ± SEM for n = 6. Intergroup differences were analyzed for statistical significance using unpaired Student’s t-test and were considered to be statistically significant from Control at op<0.05, oop<0.01, ooop<0.001; and from STZ at +p<0.05, ++p<0.01, +++p<0.001.

Acute treatments Chronic treatments Acute treatments Chronic treatments Acute treatments Chronic treatments

2) Brain LPO (Fig. 2) – Diabetes raised the levels of MDA, an index of LPO, in all brain areas, with acute values being much higher than chronic ones (from +147% to +202% vs. +9% to +18%, respectively). Acute and chronic treatments with TAU or NIC reduced the increases to values ≤7% of the control values.

5) Brain GSSG (Fig. 5) – Diabetes raised the levels of GSSG in all brain areas significantly, much more under acute (by 103-241%, p<0.001 vs. controls) than under chronic conditions (by 31-49%, p≤0.01). Both TAU and NIC were very effective in lowering the increases in GSSG caused by diabetes. When given on an acute basis, TAU was somewhat more potent than NIC (+69% to +117% vs. +51% to +162%, respectively, both at p<0.001 vs. controls). However, the potency differences between TAU and NIC disappeared when these compounds were given chronically (+16% to +28%, p≤0.05 vs. +9% to +30%, p≤0.05).

8) Brain GPx activity (Fig. 8) – Diabetes lowered the GPx activity significantly in all the brain areas examined (by 34-58% in acute studies; by 34-59% in chronic studies, p≤0.01 vs. control values). Both TAU and NIC exerted a significant attenuating effect on these decreases, with their effects being about equivalent under acute conditions (- 8 to -48% with TAU, -18 to -51% with NIC). However, under chronic treatment conditions, NIC was slightly better (-32% to +13%) than TAU (-6% to -52%)..

Acute treatments Chronic treatments Acute treatments Chronic treatments Acute treatments Chronic treatments

3) Brain NO (Fig. 3) - Diabetes raised the levels of NO in all brain areas significantly , with most acute values ((+68% to +313%, p<0.001 vs. controls) generally being higher than chronic ones (+75% to +142%, p<0.001 vs. controls). Acute treatments with TAU and NIC reduced the increases in NO due to diabetes to values below the control ones. A chronic treatment with TAU reduced the NO elevations cause by diabetes significantly but to a lesser extent than one with NIC (increases of 36-89% vs. increases of ≤25%).

6) Brain GSH/GSSG ratio (Fig. 6) – Diabetes lowered the GSH/GSSG ratio to a significant extent in all the brain areas examined (by 79-89% in acute studies; by 53-69% in chronic studies, both at p<0.001 vs. controls). Acute treatments with TAU and NIC attenuated these decreases to about similar extents (decreases of 53-72% with TAU, of 56-69% with NIC, both at p<0.001 vs. controls). When given chronically, TAU or NIC was found more effective than an acute one in sparing the GSH/GSSG ratio, with their potencies being rather similar (decreases of only 24-48% with TAU, of only 20-39% with NIC, ≤p<0.01 vs. controls).

9) Brain SOD activity (Fig. 9) – Compared to control values, diabetes lowered the SOD activity significantly in all the brain areas examined, with the decreases being greater under acute (by 55-68%, p<0.001 ) than under chronic (by 32-59%, p≤0.01) conditions. Both TAU and NIC were very effective in attenuating these losses. While acute treatments with TAU and NIC were about equal in preventing the losses in SOD activity by diabetes (-36 to -41% vs. -36 to -45%, respectively, p≤0.01), NIC was more protective than TAU after chronic treatments (-3 to -26% vs. -23 to -43%, respectively, p≤0.05).

Acute treatments Chronic treatments Acute treatments Chronic treatments

4) Brain GSH (Fig. 4) – Diabetes lowered the levels of GSH in all brain areas significantly, more under acute conditions (by 68-313%, p<0.001 vs. control), than under chronic conditions (by 75-142%, p<0.001 vs. controls). Both TAU and NIC attenuated the GSH losses caused by diabetes, with TAU appearing more effective than NIC under acute conditions (losses of 6-33% with TAU, losses of 36-56% with NIC). These protective actions were enhanced when TAU and NIC were given chronically, with the potencies being rather similar (losses ≤17% with TAU vs. losses ≤20% with NIC).

7) Brain CAT activity (Fig. 7) - Diabetes lowered the CAT activity significantly in all the brain areas examined, with the effect being greater under acute (decreases of 61-69% vs. decreases of 45-50%, respectively, all at p<0.001 vs. corresponding control values). Acute treatments with TAU or NIC attenuated these decreases to about the same extent (15-49% with TAU, 32-46% with NIC, both at p≤0.01 vs. controls), effects that were greater when these compounds were given chronically (values of -44% to +17% with TAU, -20% to +17% with NIC, p≤0.05 vs. controls).

Conclusions

♦ The treatment of rats with a diabetogen like STZ is found to induce oxidative stress throughout the CNS and marked hyperglycemia as early as 24 hr post treatment.

♦ The extent of the differences in values for indices of oxidative stress between acute and chronic treatments with NIC and TAU were marked for nonenzymatic ones and rather close for enzymatic ones.

♦ Although the changes in biochemical indicators of oxidative stress were, for the most part, significant, their extents were not consistent from area to area or with the degree of hyperglycemia.

♦ Both TAU and NIC were not only very effective in counteracting diabetes-induced oxidative stress in the CNS but also rather similar in their potencies.

References Aebi H (1984) Meth Enzymol 105:121-126. Akerboom TP and Sies H (1981) Meth Enzymol 77:373-382. Biasetti M and Dawson Jr. R (2002) Amino Acids 22:351-368. Buege JA and Aust SD (1978) Meth Enzymol 53:302-310. Cetin F, Dincer S, Ay R and Guney S (2012) Afr J Pharm Pharmacol 6:1099-1105. Fox JB, Zell TE and Wasserman AE (1981) J Assoc Off Anal Chem 64:1397-1402. Günzler A and Flohé L (1985) in CRC Handbook of Methods of Oxygen Radical Research, Greenwald RA (Ed.), CRC Press, Boca Raton, FL, pp. 285-290. Guntherberg H and Rost J (1966) Anal Biochem 15:205-210. Kolb H and Burkart V (1999) Diabetes Care 22 (Suppl 2):B16-B20. Lenzen S (2008) Diabetologia 51:216-

226. Kamatat JP and Devasagayam TP (2012) Redox Rep 4:179-184. Misra HP (1985) in Handbook of Methods of Oxygen Radical Research, Greenwald RA (Ed.), CRC Press, Boca Raton , FL, pp. 234-241. Pandya KG, Patel MR and Lau-Cam CA (2000) J Biomed Sci 17(Suppl 1):S16. Raabo E and Terkildsen TC (1960) Scand J Clin Lab Invest 12:4:402-407. Stevens MJ, Li F, Drel VR et al. (2007) J Pharmacol Exp Ther 320:458-464. Shaffer SW, Azuma J, Mozaffari M (2009) Can J Physiol Pharmacol 87:91-99. Syrdah MM (2014) Diabetes Met Syndr Clin Res Rev, http://dx.doi.org/10.1016/j.dsx.2014.5.001 Yamada K, Nonaka K, Hanafusa T et al. (1982) Diabetes 31:9749-753.

1) Plasma glucose (Fig. 1) – The treatment of rats with STZ (60 mg/kg, i.p.) caused a marked increase in plasma glucose in both the acute (+117%, p<0.001) and chronic (+254%, p<0.001) studies relative to control rats. While neither NIC nor TAU were able to attenuate the acute diabetic plasma glucose level to a significant extent (only 9% and 5% reductions, respectively), they were significantly effective when administered on a chronic daily basis, in which case the diabetic plasma glucose was reduced by 31% (p<0.01) by NIC and by 21% (p<0.05) by TAU.