Toxicology and Applied Pharmacology 258 (2012) 296–308

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Toxicology and Applied Pharmacology

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Taurine exerts hypoglycemic effect in alloxan-induced diabetic rats, improvesinsulin-mediated glucose transport signaling pathway in heart and amelioratescardiac oxidative stress and apoptosis

Joydeep Das, Vandana Vasan, Parames C. Sil ⁎Division of Molecular Medicine, Bose Institute P-1/12, CIT Scheme VII M, Kolkata-700054, India

Abbreviations: ALX, alloxan; CAT, catalase; DAB, 3drochloride; EtBr, ethidium bromide; FACS, fluoresGLUT4, glucose transporter type 4; GSH, glutathioneGST, glutathione S-transferase; GR, glutathione reductatein; IL-6, inter leukin6; IR, insulin receptor; IRS1, insulactate dehydrogenase; LDL, low-density lipoprotein; Mnuclear factor kappa B; ROS, reactive oxygen speciesSTE buffer, sodium chloride-tris-EDTA buffer; TAU, tauritor alpha; TUNEL, terminal transferase mediated dUTP n⁎ Corresponding author at: Division of Molecular Med

Scheme VII M, Calcutta-700054, West Bengal, India. FaxE-mail addresses: parames@bosemain.boseinst.ac.in

(P.C. Sil).

0041-008X/$ – see front matter © 2011 Elsevier Inc. Alldoi:10.1016/j.taap.2011.11.009

a b s t r a c t

a r t i c l e i n f o

Article history:Received 27 September 2011Revised 3 November 2011Accepted 15 November 2011Available online 26 November 2011

Keywords:AktAlloxan-induced diabetesApoptosisCytokinesGLUT4HeartHyperglycemiaInsulin receptorTaurine

Hyperlipidemia, inflammation and altered antioxidant profiles are the usual complications in diabetesmellitus. In the present study, we investigated the therapeutic potential of taurine in diabetes associatedcardiac complications using a rat model. Rats were made diabetic by alloxan (ALX) (single i.p. dose of 120 mg/kgbody weight) and left untreated or treated with taurine (1% w/v, orally, in water) for three weeks eitherfrom the day of ALX exposure or after the onset of diabetes. Animals were euthanized after three weeks.ALX-induced diabetes decreased body weight, increased glucose level, decreased insulin content, enhancedthe levels of cardiac damage markers and altered lipid profile in the plasma. Moreover, it increasedoxidative stress (decreased antioxidant enzyme activities and GSH/GSSG ratio, increased xanthine oxi-dase enzyme activity, lipid peroxidation, protein carbonylation and ROS generation) and enhanced theproinflammatory cytokines levels, activity of myeloperoxidase and nuclear translocation of NFκB in thecardiac tissue of the experimental animals. Taurine treatment could, however, result to a decrease in the elevatedblood glucose and proinflammatory cytokine levels, diabetes-evoked oxidative stress, lipid profiles and NFκBtranslocation. In addition, taurine increased GLUT 4 translocation to the cardiac membrane by enhancedphosphorylation of IR and IRS1 at tyrosine and Akt at serine residue in the heart. Results also suggest that taurinecould protect cardiac tissue from ALX induced apoptosis via the regulation of Bcl2 family and caspase 9/3proteins. Taken together, taurine supplementation in regular diet could play a beneficial role in regulatingdiabetes and its associated complications in the heart.

© 2011 Elsevier Inc. All rights reserved.

Introduction

Diabetes is the most common and serious metabolic disease.Diabetes is mainly found to be of two types. In type 1, which isalso known as IDDM or insulin dependent diabetes mellitus, insu-lin is produced in lesser amount. In type 2, also known as NIDDMor noninsulin dependent diabetes mellitus, the pancreas is usuallyproducing enough insulin, but for unknown reasons the body cannot

,3′-diaminobenzidine tetrahy-cence activated cell sorting;; GSSG, glutathione disulfide;se; HDL, high-density lipopro-lin receptor substrate 1; LDH,DA, malondialdehyde; NF-κB,; SOD, superoxide dismutase;ne; TNF-α, tumor necrosis fac-ick end-labeling.icine, Bose Institute P-1/12, CIT: +91 33 2355 3886., parames_95@yahoo.co.in

rights reserved.

use the insulin effectively, a condition called insulin resistance. Thechronic disorders related to diabetes apart from hyperglycemia andhyperlipidemia are cardiovascular complications, nephropathy andretina damage (Baynes and Thorpe, 1999; Ceriello, 2000; Yim et al.,2007).

The major role of insulin in diabetes is to maintain whole bodyglucose homeostasis via glucose transporter 4 (GLUT4), expressedin adipose tissue, skeletal and cardiac muscles (Charron et al., 1999;Haruta et al., 1995). During insulin stimulation, intracellular vesiclesthat store GLUT4, translocate to the plasma membrane and facilitateglucose uptake (Bryant et al., 2002; Pessin et al., 1999). Under dia-betic condition, reduced expression of GLUT4 causes impairment ofinsulin signaling and stimulates glucose production in the liver.These alterations lead to high glucose concentrations in blood(Nizamutdinova et al., 2009).

Hyperglycemia-induced generation of free radicals (oxidativestress) contributes to the development and progression of diabetesand other related complications. Therefore, an agent that possessesboth hypoglycemic and antioxidant activities would be a therapeutictool for diabetic patients (Manna et al., 2010a,b). Taurine, a sulfurcontaining beta amino acid, is present in most animal tissues and is

297J. Das et al. / Toxicology and Applied Pharmacology 258 (2012) 296–308

essential for the normal functioning of different organs (Brosnan andBrosnan, 2006). Taurine exhibits antioxidative properties, membranestabilizing effect, regulates intracellular Ca2+ concentration, inhibitsapoptosis, reduces the levels of pro-inflammatory cytokines in vari-ous organs and controls blood pressure (Aerts and Van Assche,2002; Das et al., 2008, 2009a,b, 2010a–c, 2011a,b; Kontny et al.,2000; Manna et al., 2008a,b, 2009; Racasan et al., 2004; Sinha et al.,2007, 2008a,b). Taurine is found at high concentrations inside gluca-gon and somatostatin-containing cells in the pancreatic islets andincreases insulin secretion, sensitivity and glucose uptake (Cherifet al., 1998; De la Puerta et al., 2010; Kaplan et al., 2004) in differentexperimental conditions. Literature suggests a considerable varia-tion about the opinion on the mode of action of taurine in diabetes.Kulakowski and Maturos (1984) reported that the hypoglycemiceffect of taurine was not mediated via increased insulin release; onthe other hand, Pandya et al. (2010) and Chang and Kwon (2000)reported that taurine increased insulin secretion in streptozotocin-induced diabetic animals. Besides, Brons et al. (2004) reported thattaurine supplementation had no effect on insulin secretion andglucose level in prediabetic human. The authors, however, alsoexpressed the opinion that they could not rule out the possibilityof hypoglycemic effect of taurine if diabetic subjects had beentaken (as reported by Elizarova and Nedosugova (1996)).

The beneficial effects of taurine in diabetes mellitus and its cardio-protective role in other pathophysiological conditions have beenknown (Das et al., 2011a; Gavrovskaya et al., 2008; Ghosh et al., 2009;Winiarska et al., 2009), although the exact mechanism of hypoglycemicaction of taurine in cardiac associated complications is not properlydefined. The aim of this present study has, therefore, been set to inves-tigate the mechanisms of possible beneficial action of taurine againstALX (2,4,5,6-tetraoxypyrimidine; 2,4,5,6-pyrimidinetetrone)-induceddiabetes and its associated complications in cardiac tissue of rats. Inthe present study, attempts have, therefore, been made to investigatethe mechanisms of hypoglycemic, antioxidant, antiinflammatory andantiapoptotic action of taurine in ALX-induced diabetic heart. Theoutcome, we believe, might provide important information about itsusefulness as a therapeutic agent for the treatment of diabetes andrelated cardiac dysfunctions.

Materials and methods

Chemicals

Taurine (2-aminoethane sulfonic acid), alloxan, bovine serum al-bumin (BSA), Bradford reagent, anti Bcl 2, anti Bcl XL, anti caspase3, anti Akt, and anti NFkB antibodies were purchased from Sigma-Aldrich Chemical Company (St. Louis, MO, USA). Other antibodies,like, anti IR, anti IRS1, and anti GLUT4 were purchased from Abcam(Cambridge, Cambridgeshire, UK). Kits for measurement of bloodglucose, LDH, uric acid and total cholesterol were purchased fromSpan Diagnostic Ltd., Surat, Gujarat, India. All other chemicals werebought from Sisco Research Laboratory, Andheri, Mumbai, India.TUNEL assay kit was purchased from Invitrogen, Eugene, Oregon,USA.

Animals

Adult male Wistar rats weighing approximately 160–180 g werepurchased from M/S Gosh Enterprises, Kolkota, India. Animals wereacclimatized under laboratory conditions for two weeks prior toexperiments. All the experiments with animals were carried outaccording to the guidelines of the institutional animal ethical com-mittee (IAEC), Bose Institute, Kolkata and full details of the studywere approved by both IAEC and CPCSEA (committee for the purposeof control and supervision on experiments on animals), Ministry ofEnvironment and Forests, New Delhi, India.

Experimental design for in vivo treatments

Experimental design needed for the present in vivo study has beensummarized (in Fig. 1) as follows: Forty-five rats were randomlyassigned to five groups.

Group 1—Normal group: Six rats received neither ALX nor taurineand received only water as vehicle. No mortality was found.Group 2—TAU group: Six rats received only 1% taurine (w/v inwater, orally). No mortality was found.Group 3—ALX group: Thirteen rats received single dose of ALX(Verma et al., 2010) at a dose of 120 mg/kg body wt in citratebuffer, pH 4.5, i.p. After 3 days of ALX injection, rats having bloodglucose level in excess of 300 mg/dL were considered as diabetic.Six rats died during the experimental period.Group 4—ALX&TAU simultaneous treatment group: Ten rats re-ceived taurine (1% w/v in water, orally, 1 h before ALX injection)from the day on which ALX was injected for 21 days. Two ratsdied during the experimental period.Group 5—ALX+TAU post treatment group: Ten rats receivedtaurine (1% w/v in water, orally) from the 4th day after ALXinjection for 21 days. Three rats died during the experimentalperiod.

Collection of blood, pancreas and heart

Rats in each group were bled every 3 days from the lateral vein ofthe tail and 100 μL blood was taken for the measurement of plasmaglucose.

The experimental rats were euthanized under light ether anesthe-sia after 3 weeks of treatment with taurine. Pancreases and heartswere removed. Hearts were either stored at −80 °C till biochemicalanalysis later or fixed in 10% buffered formalin for TUNEL and histo-logical assessments. Pancreases were fixed in 10% buffered formalinfor only histological assessments. The body weight and heart weightwere measured and compared between groups. Blood samples weredrawn from the caudal vena cava. Blood was collected in test tubescontaining heparin solution and centrifuged at 1500×g for 10 minto obtain plasma. The plasma was immediately stored at −80 °Cuntil use.

Preparation of nuclear, mitochondrial, cytosolic and membrane fractions

The hearts were minced, washed with saline buffer and homoge-nized in a Dounce glass homogenizer in homogenizing buffer (50 mMphosphate buffer/1 mM EDTA, pH 7.5, containing 1.5 mM MgCl2,10 mM KCl, and supplemented with protease and phosphatase inhibi-tors). The homogenates were spun down for 10 min at 500×g at 4 °C.The supernatant was collected and recentrifuged at 2000×g for10 min. The pellet was resuspended in the same buffer and taken asnuclear fraction and stored at −80 °C as described by the method ofLizotte et al. (2009). The supernatant was recentrifuged at 12,000×gfor 10 min at 4 °C, and pellet was resuspended in 200 mM mannitol,50 mM sucrose, 10 mmol/L Hepes-KOH (pH 7.4) and stored as mito-chondrial fraction at −80 °C as described by the method of Jang et al.(2004) with some modifications. The final supernatant was taken andcentrifuged for 1 h at 40,000×g. The resultant supernatant was usedas cytosolic fraction and stored at 4 °C. The pellet was resuspended inthe above-mentioned homogenizing buffer, containing 1% (v/v) TritonX-100, by sonication for 7×1 s cycles and again centrifuged for 1 h at40,000×g. The supernatant now contained the extracted membranefraction and stored at 4 °C (Huisamen et al., 2001).

In the present study, the nuclear fraction has been used for thewestern blot analysis of NFκB; membrane fraction has been used for

Male Wistarrats

Taurine(-)

ALX (-)Normal group

n = 6

ALX (+)120 mg/kg i.p.,

Single dose, Day 1ALX group

n = 6

Taurine (+)1% (w/v) in water, Orally, Day 1 -21

ALX (-) TAU g roupn = 6

ALX (+)120 mg/kg i.p.,

Single dose, Day 1

ALX+TAU post group , n = 6

Sacrificed after 21 days

Taurine (+)1% (w/v) in water, Orally, Day 4 -24

ALX&TAU simultaneous group , n = 6

ALX (+)120 mg/kg i.p.,

Single dose, Day 1Sacrificed after

24 days

Fig. 1. Schematic diagram of the experimental protocol. “n=6” indicates the numbers of alive rats after 21 days of treatment.

298 J. Das et al. / Toxicology and Applied Pharmacology 258 (2012) 296–308

the western blot analysis of GLUT4; mitochondrial fraction has beenused for the western blot analysis of cytochrome c and membranepotential determination and finally cytosolic fraction has been usedfor all other western blot analyses, determination of MDA, proteincarbonyl, thiol based antioxidants, xanthine oxidase, ROS, antioxidantenzymes activities, myeloperoxidase activity, IL-6 and TNF-α levels.

Determination of protein content

The protein content of the experimental samples was measuredby the method of Bradford (1976) using crystalline BSA as standard.

Biochemical analyses

Plasma glucose and specific markers related to cardiac dysfunctionand lipid profiles e.g. LDH, uric acids, total cholesterol and triglyceridelevels in the plasma were estimated by using standard kits purchasedfrom Span Diagnostic Ltd., Surat, Gujarat, India. The lipid peroxida-tion, protein carbonyl content and intracellular ROS were estimatedfollowing the method as described by Das et al. (2009a). Activitiesof antioxidant enzymes (SOD, CAT, GST, GR and GPx) and cellularmetabolites levels (GSH and GSSG) in the heart tissue were deter-mined following the method as described by Das et al. (2009b).Myeloperoxidase activity was measured by the methods of Naitoet al. (1998).

Determination of xanthine oxidase activity in cardiac tissue

Xanthine oxidase activity was assessed by measuring the enzy-matic oxidation of xanthine to uric acid following the methods ofBergmeyer et al. (1974). The reaction mixture (3 mL) contained1.9 mL of 50 mM potassium phosphate buffer, pH 7.5 and 1 mL of0.15 mM xanthine. The mixture was mixed by inversion and equili-brated at 25 °C. The absorbance was measured at 290 nm until aconstant value was obtained. The reaction was started by adding100 μL of tissue extract and the increase in absorbance wasmeasuredat 290 nm for 4 min at 25 °C.

Determination of plasma insulin and cardiac TNF-α and IL-6 levels byELISA

ELISA was performed by the method of Ausubel et al. (2003). 50 μLantibodies at a concentration of 5 μg/mL were loaded in 96 well platesand incubated at 4 °C overnight. After that, the well plates werewashed with wash buffer (25 mM Tris, pH 8.0, 150 mM NaCl and

0.5% Tween 20) twice to remove unbound antibodies. Then 50 μL ofplasma/cytosolic protein was added into the wells and incubated at4 °C overnight. After that, the well plates were washed with washbuffer. Then 50 μL HRP conjugated secondary antibody (200 ng/mL)was added and incubated for 2 h at 4 °C. Then again washing withwash buffer was repeated. The color was developed by adding100 μL of TMB/H2O2 solution and reaction was stopped by adding2 N H2SO4. Then absorbance was measured at 450 nm.

Histological studies

Pancreases and hearts from the normal and experimental ratswere fixed in 10% buffered formalin and were processed for paraffinsectioning. Sections of about 5 μm thickness were stained with he-matoxylin and eosin to evaluate under light microscope.

Determination of mitochondrial membrane potential (Δψm)

Analytic flow cytometric measurements for the membrane poten-tial (Δψm) of isolated mitochondria were performed using a FACScanflow cytometer with an argon laser excitation at 488 nm and a525 nm band pass filter as described by the method of Das et al.(2011a). Mitochondrial membrane potential (Δψm) was measuredon the basis of cell retention of the fluorescent cationic probe rhoda-mine 123.

Immunoblotting in cardiac tissue

Immunoblotting was performed as described by the method ofDas et al. (2011a). An equal amount of protein (50 μg) from eachsample was resolved by 10% SDS-PAGE and transferred to PVDFmembrane. Membranes were blocked at room temperature for 2 hin blocking buffer containing 5% non-fat dry milk to prevent non-specific binding and then incubated with primary antibodies sepa-rately at 4 °C overnight; namely, anti-NF-κB (p65) (1:250 dilution,for nuclear fraction), anti GLUT4 (1:1000 dilution, for membranefraction), anti p-IR (1:1000 dilution), anti p-IRS1 (1:1000 dilution),anti Bad (1:1000 dilution), anti Bax (1:1000 dilution), anti Bcl-2(1:1000 dilution), anti Bcl-xL (1:1000 dilution), anti caspase3(1:1000 dilution) and anti Akt (1:100 dilution) for cytosolic fractionand anti cytochrome c (1:1000 dilution) for both cytosolic andmitochondrial fractions. The membranes were washed in TBST(50 mmol/L Tris–HCl, pH 7.6, 150 mmol/L NaCl, 0.1% Tween 20)for 30 min and incubated with appropriate HRP conjugated sec-ondary antibody (1:2000 dilution) for 2 h at room temperature

-3 0 3 6 9 12 15 18 21 24

100

200

300

400

500

abc***,d*

abc***,d**

abcd***abcd***

ab***,c**abc***

abc***

abc***abc***

abc***abc***

abc***

abd***abd***abd***

abc***,d*

ab***ab***ab***ab***ab***ab***ab***ab***

ALX+TAUALX&TAU

ALX

NormalTaurine

Pla

sma

gluc

ose

(mg/

dl)

(days)

Fig. 2. Time dependent effect of taurine and diabetes on plasma glucose levels up to3 weeks. Normal: plasma glucose levels in normal rats; taurine: plasma glucose levelsin rats treated with only taurine (1%) for 3 weeks; ALX: plasma glucose levels in rats(closed square) received ALX in a single dose (120 mg/kg body weight, i.p.); ALX&TAUsimultaneous (empty circle) and ALX+TAU post (closed circle): plasma glucose levelsin diabetic rats treated with taurine. Data represent the average±SD of 6 separateexperiments in each group. “a” indicates that it differs significantly from normal;“b” indicates that it differs significantly from TAU; “c” indicates that it differs signifi-cantly from ALX; “d” indicates that it differs significantly from ALX&TAU. ⁎Pb0.05,⁎⁎Pb0.01, and ⁎⁎⁎Pb0.001.

299J. Das et al. / Toxicology and Applied Pharmacology 258 (2012) 296–308

and developed by the HRP substrate 3,3′-diaminobenzidine tetra-hydrochloride (DAB) system (Bangalore Genei, India).

DNA fragmentation assay

Cardiac tissue was washed with STE buffer (0.1 M NaCl, 10 mMTris–HCl, 1 mM EDTA, pH 8) and 1 mL homogenization buffer(0.1 M NaCl, 0.2 M sucrose, 0.01 M EDTA, 0.3 M tris, pH 8) and100 μL 10% SDS was added and mixed by vortexing. The mixturewas incubated at 65 °C for 1 h. 175 μL of 8(M) potassium acetatewas then added and incubated in ice for 1 h, centrifuged and the su-pernatant was collected. Equal volume of phenol–chloroformmixturewas added, mixed and centrifuged to separate phases. The upper-most layer was taken in a fresh tube. Equal volume of chloroformwas added and centrifuged. The aqueous layer was taken in a freshtube. 1/10th vol of 3(M) sodium acetate (pH 7.4) and 2.5 times volof ethanol were added and centrifuged. The precipitated DNA waswashed with 80% ethanol.

The DNA fragmentation has been assayed by electrophoresinggenomic DNA samples, isolated as described above from normal aswell as experimental rat hearts on agarose/EtBr gel.

TUNEL staining

Paraffin embedded cardiac tissue sections (5 μm) were warmed30 min (64 °C), deparaffinized and rehydrated. Terminal transferasemediated dUTP nick end-labeling of nuclei was performed by usingAPO-BrdU TUNEL Assay kit (A-23210; Molecular Probes, Eugene,OR) following the manufacturer's protocol.

Statistical analysis

All the values are expressed as mean±S.D. (n=6). Significantdifferences between the groups were determined with SPSS 10.0software (SPSS Inc., Chicago, IL, USA) for Windows using one-wayanalysis of variance (ANOVA) and the group means were comparedby Tukey's test. A difference was considered significant at thepb0.05 level.

Results

Taurine suppressed the change in body weight, plasma glucose, insulinand cardiotoxicity

Approximately 85% of the rats injected with ALX developed exper-imental IDDM, which were characterized by significant body weight(Pb0.001) and heart weight (Pb0.01) loss (Table 1), increased plas-ma glucose (Pb0.001) (Fig. 2) and decreased plasma insulin levels(Pb0.001) (Fig. 3). The rats were behaviorally inactive and mortalityrate was approximately 50%. However, treatment with taurine (1%)

Table 1Effect of alloxan and taurine on the heart weight, body weight and levels of the serum mar

Parameters Normal control TAU treated

Body weight (g) 202.4±7.12 202.2±7.58Heart weight (g) 0.58±0.023 0.61±0.115Ratio of the heart weight to the body weight (%) 0.28±0.010 0.31±0.013LDH (U/L) 131.66±4.58 116.47±3.82a,⁎

Uric acid (mg/dL) 5.166±0.22 5.868±0.27

Values are expressed as mean±SD, for 6 animals in each group.a Indicates that it differs significantly from normal.b Indicates that it differs significantly from TAU.c Indicates that it differs significantly from ALX.

⁎ Pb0.05.⁎⁎ Pb0.01.⁎⁎⁎ Pb0.001).

for 3 weeks recovered the loss of body weight (Pb0.001) and heartweight (Pb0.01) and increased the survival rate (70–80%) causedby ALX. Taurine treatment also decreased the plasma glucose(Pb0.001) and increased the plasma insulin levels (Pb0.001, in caseof taurine simultaneous treated groups and Pb0.01, in case of taurinepost treated groups) in diabetic rats. A significant difference wasdetected in plasma glucose level between taurine simultaneous andtaurine post treated groups after three weeks of treatment(Pb0.05). Plasma insulin levels were different between normal andtaurine simultaneous treated groups (Pb0.05) as well as normaland taurine post treated groups (Pb0.01).

Results showed that under diabetic condition, the plasma levels ofthe cardiac damage markers, e.g. LDH (Pb0.001) and uric acid(Pb0.05) (Table 1) were significantly increased compared to normaland only taurine treated rats. However taurine treatment effectivelydecreased the LDH (Pb0.001) and uric acid (Pb0.05) levels comparedto diabetic rats.

Taurine normalized the ALX-induced damage of pancreas

Histological assessments of various pancreatic segments of thenormal and experimental rats were presented in Fig. 4. The pancreatic

kers related to cardiac dysfunction of experimental animals.

ALX treated ALX&TAU ALX+TAU

143.6±5.18a,⁎⁎⁎,b,⁎⁎⁎ 198.8±6.94c,⁎⁎⁎ 197.0±6.91c,⁎⁎⁎

0.47±0.017a,⁎⁎,b,⁎⁎ 0.59±0.012c,⁎⁎ 0.57±0.018c,⁎⁎

0.32±0.015 0.32±0.012 0.28±0.012199.18±7.96a,⁎⁎⁎,b,⁎⁎⁎ 153.83±5.69a,⁎⁎,b,⁎⁎⁎,c,⁎⁎⁎ 162.83±6.14a,⁎⁎,b,⁎⁎⁎,c,⁎⁎⁎

10.404±0.32a,⁎,b,⁎ 5.424±0.17c,⁎ 5.434±0.17c,⁎

i

0

10

20

30

40

50

a*,b**,c***

a**,b**,c**

ab***

TAU

ALX&TAU

ALX+TAUALX

Seru

m in

sulin

( μm

ol/m

l)

NOR

Fig. 3. Effect of taurine and diabetes on plasma insulin levels after 3 weeks of treat-ment. Each column represents mean±SD, n=6. “a” indicates that it differs signifi-cantly from normal; “b” indicates that it differs significantly from TAU; “c” indicatesthat it differs significantly from ALX. ⁎Pb0.05, ⁎⁎Pb0.01, and ⁎⁎⁎Pb0.001.

300 J. Das et al. / Toxicology and Applied Pharmacology 258 (2012) 296–308

islets of Langerhans were smaller with decreased number of β-cellsin the ALX-induced diabetic rats than the normal group. However,treatment with taurine prevented the development of ALX-inducedchanges, thereby suggesting the protective role of taurine in thepancreas of diabetic rats.

Taurine suppressed ALX-induced oxidative stress

In our present study, the occurrence of oxidative stress in ALX-induced diabetic rat heart was tested by measuring lipid peroxidation

Fig. 4. Effect of taurine on histopathological changes in pancreatic tissue (stained withhematoxylin and eosin dye). Normal: pancreatic section of normal rat pancreas(×200); ALX: pancreatic section from the diabetic group (×200); ALX&TAU: pancreatictissue section from the taurine simultaneous and ALX+TAU: taurine post treated ratsrespectively (×200). Arrows indicate islets of Langerhans.

(MDA), protein carbonylation, (Table 2) status of intracellular thiols(GSH, GSSG), intracellular ROS, xanthine oxidase (Table 3) and anti-oxidant enzymes activities (Table 4). We observed that lipid peroxi-dation (Pb0.001), protein carbonylation (Pb0.01), intracellular ROSlevel (Pb0.001) and xanthine oxidase activity (Pb0.01) were in-creased in the heart of diabetic rats compared to normal rats. Onthe other hand cardiac GSH:GSSG ratio (Pb0.001) and anti-oxidantenzymes activities, e.g. SOD (Pb0.001), CAT (Pb0.001), GST (Pb0.01),GR (Pb0.001) and GPx (Pb0.001) were decreased compared to normalrats. However, the decrease of intracellular GSH content was notsignificant, whereas diabetes decreased the GSH/GSSG ratio as aresult of increased GSSG (Pb0.05) content in diabetic rats. Allthese clearly reflected the involvement of oxidative stress in thispathophysiological condition. However, treatment with taurinesignificantly decreased lipid peroxidation (Pb0.001), protein car-bonylation (Pb0.01), ROS level (Pb0.001), and xanthine oxidaseactivity (Pb0.01) and increased GSH:GSSG ratio (Pb0.001) andanti-oxidant enzymes activities; SOD (Pb0.001), CAT (Pb0.001),GST (Pb0.01), GR (Pb0.001) and GPx (Pb0.001) compared to ALX-exposed group and thereby proving it to be an effective anti-oxidantin this regard.

Taurine normalized the altered lipid profile in diabetic rats

Table 5 showed the effect of taurine on plasma lipid profile indiabetic rats. Under hyperglycemic condition plasma triglyceride(Pb0.001), total cholesterol (Pb0.001), LDL (Pb0.001), LDL/HDL(Pb0.001) and total cholesterol/HDL (Pb0.001) ratios were increasedsignificantly. Taurine significantly lowered the ALX-induced increasedtriglyceride (Pb0.001), total cholesterol (Pb0.001) and LDL (Pb0.001)levels. However, HDL level was not changed significantly in diabeticrats. Taurine also showed significant decrease in LDL/HDL (Pb0.001)and total cholesterol/HDL (Pb0.001) ratios in diabetic rats. All theseresults suggested that taurine is effective in maintaining the lipidprofile near to that of control in this animal model of diabetes.

Taurine effectively reduced ALX-induced cardiac damage

Fig. 5 showed the histological assessments of cardiac segments ofnormal, ALX-exposed and taurine-treated animals. Disorganization ofthe normal radiating pattern of cell plates in the ALX-injected rathearts was observed. However, treatment with taurine reducedsuch changes in ALX-injected rat hearts.

Taurine reduced the diabetes-associated up-regulation of NFkB, IL-6,TNF-α and myeloperoxidase activity

NF-κB regulates a number of genes (including those coding for keyinflammatory cytokines, like IL-6, TNF-α etc.) involved in inflamma-tion. We observed that the protein level of p65 sub-unit of NF-κB innuclear extracts and cardiac levels of the pro-inflammatory cytokines,namely IL-6 (Pb0.001) and TNF-α (Pb0.001) was markedly elevatedin diabetic rats compared to normal rats (Fig. 6). Our results alsoshowed that the activity of myeloperoxidase was also increased indiabetic rats (Pb0.001) compared to normal rats (Fig. 4). However,treatment with taurine significantly decreased NF-κB expression,IL-6 (Pb0.001) and TNF-α (Pb0.001) levels and myeloperoxidaseactivity (Pb0.001) compared to diabetic rats. All these resultssuggested that taurine effectively worked as an anti-inflammatoryagent under diabetic conditions.

Taurine improved insulin signaling in cardiac tissue of diabetic rats

It is well known that glucose uptake is controlled by insulin-mediated GLUT4 translocation to the plasma membrane. Therefore,we have investigated whether the hypoglycemic effect of taurine

Table 2Effect of alloxan and taurine on the status of the lipid peroxidation and protein carbonylation.

Parameters Normal control TAU treated ALX treated ALX&TAU ALX+TAU

MDA (nmol/mg protein) 1.123±0.03 1.083±0.03 4.363±0.09a,⁎⁎⁎,b,⁎⁎⁎ 2.166±0.06a,⁎⁎,b,⁎⁎,c,⁎⁎⁎ 2.178±0.06a,⁎⁎,b,⁎⁎,c,⁎⁎⁎

Protein carbonylation (nmol/mg protein) 14.27±0.614 11.37±0.469a,⁎⁎ 20.02±0.099a,⁎⁎,b,⁎⁎⁎ 14.72±0.614b,⁎⁎,c,⁎⁎ 13.45±0.573b,⁎,c,⁎⁎

Values are expressed as mean±SD, for 6 animals in each group.a Indicates that it differs significantly from normal.b Indicates that it differs significantly from TAU.c Indicates that it differs significantly from ALX.

⁎ Pb0.05.⁎⁎ Pb0.01.⁎⁎⁎ Pb0.001.

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was mediated via the insulin-dependent regulation of myocardialGLUT4 translocation and subsequent glucose uptake in ALX-induceddiabetes. We observed that tyrosine phosphorylation of IR, IRS1,serine phosphorylation of Akt and GLUT4 expression at the mem-brane fraction were decreased in the heart of diabetic rats, indicatingless glucose uptake (Fig. 7). However, taurine treatment significantlydecreased the ALX-induced reduction of phosphorylation of IR,IRS1, and Akt and increased the membrane translocation of GLUT4.These results showed that taurine enhanced insulin sensitivity inALX-induced diabetic rats.

Taurine protected against ALX-induced apoptosis in heart

Next we have investigated the mode of cell death in ALX-induceddiabetic heart. We observed that ALX significantly increased thepro-apoptotic Bad/Bax and decreased anti-apoptotic Bcl-2/Bcl-xLprotein expression, in other words, it disturbed the balance betweenthe pro- and anti-apoptotic Bcl-2 family protein expressions. Inaddition, ALX also reduced the mitochondrial membrane potentialand increased the translocation of cytochrome c from mitochondria(reduced protein expression) into cytosol (increased protein ex-pression) and protein expressions of cleaved caspase 9/3 (Fig. 8).ALX-induced apoptosis was also evident from the ladder pattern(hallmark of apoptosis) obtained in DNA gel electrophoresis(Fig. 8) and TUNEL assay (Fig. 9). As shown in Fig. 9, TUNEL positivenuclear staining was observed in ALX-induced diabetic rat heartindicating apoptosis of the cardiac cells. However, taurine effective-ly reduced the ALX-induced disturbances in the regulations of Bcl-2family proteins, cytosolic translocation of cytochrome c, cleavage ofcaspase 9/3 and number of TUNEL positive nucleus. On the otherhand, taurine increased mitochondrial membrane potential andDNA integrity in ALX-exposed rats. All these results clearly demon-strated the anti-apoptotic role of taurine in cardiac tissue underALX-induced diabetic conditions.

Table 3Effect of alloxan and taurine on the status of the thiol based antioxidants, xanthine oxidase

Parameters Normal control TAU treated

GSH (nmol/mg protein) 18.68±0.734 18.44±0.722GSSG (nmol/mg protein) 0.64±0.022 0.62±0.021Redox ratio (GSH/GSSG) 29.19±1.359 29.74±1.387Xanthine oxidase activity (mU/mg protein) 0. 48±0.032 0.47±0.0265Intracellular ROS (% over control) 100±4 67±2.35

Values are expressed as mean±SD, for 6 animals in each group.a Indicates that it differs significantly from normal.b Indicates that it differs significantly from TAU.c Indicates that it differs significantly from ALX.

⁎ Pb0.05.⁎⁎ Pb0.01.⁎⁎⁎ Pb0.001.

Discussion

Diabetes mellitus is the most common metabolic syndrome andan important health problem worldwide. About 65–70% of diabeticpatients (both type 1 and type 2) die as the outcome of heart disease(El-Seweidy et al., 2011). In the present study, we investigated themechanism of hypoglycemic effect of taurine and its beneficial effectin cardiac damage under diabetic conditions as well. Taurine hasbeen administered orally through drinking water so that it could bebetter absorbed and in consequence, more effective than routedthrough added to foods (Winiarska et al., 2009).

ALX, at a dose of 120 mg/kg body weight, caused sufficient dam-age to pancreatic β cells so that secreted insulin was not enough toregulate blood glucose and resulted to a significant increase inblood glucose levels (Verma et al., 2010). In our experiment, wehave shown that taurine markedly decreased plasma glucose levelsin ALX-induced diabetic rats via the potent insulin effect by protect-ing pancreatic β cells. Diabetes is also characterized by severe lossof body weight. The decreased body weight in diabetic rats is due toexcessive breakdown of muscle tissue, fats and metabolism of pro-teins (Chatterjea and Shinde, 2002; Roy et al., 2011). Taurine treat-ment to diabetic rats, however, showed significant increase in theaverage body weights compared to the untreated diabetic rats.

Alterations of lipid profile are common in diabetic conditions(Kain et al., 2010). In diabetes, since blood glucose is not utilized bytissue, the fatty acid from adipose tissue are mobilized for energypurpose and excess fatty acids are accumulated in the liver and con-verted to triglycerides (Shih et al., 1997). Under diabetic condition,cholesterol level was increased due to decreased cholesterol absorp-tion and increased cholesterol biosynthesis (Gylling and Miettinen,1997). Chronic insulin deficiency is associated with a diminishedlevel of LDL receptor which causes the increase in LDL particles andresults to an increase in LDL-cholesterol levels in diabetes mellitus(Suryawanshi et al., 2006). Similar with other studies (Kain et al.,2010; Nizamutdinova et al., 2009), we also observed increased

activity and intracellular ROS.

ALX treated ALX&TAU ALX+TAU

16.80±0.640 17.92±0.796 17.98±0.7990.99±0.039a,⁎,b,⁎ 0.65±0.022c,⁎ 0.67±0.024c,⁎

16.92±0.746a,⁎⁎⁎,b,⁎⁎⁎ 27. 56±1.278c,⁎⁎⁎ 26.84±1.242c,⁎⁎⁎

0. 0.80±0.055a,⁎⁎,b,⁎⁎ 0.53±0.0445c,⁎⁎ 0.55±0.045c,⁎⁎

210±9.5a,⁎⁎⁎,b,⁎⁎⁎ 102±4.1b,⁎⁎,c,⁎⁎⁎ 107±4.35b,⁎⁎,c,⁎⁎⁎

Table 4Effect of alloxan and taurine on the activities of the antioxidant enzymes in cardiac tissue.

Name of the parameters Normal control TAU treated ALX treated ALX&TAU ALX+TAU

SOD (unit/mg protein) 16.8±0.74 17.4±0.67 5.2±0.16a,⁎⁎⁎,b,⁎⁎⁎ 14.8±0.64b,⁎,c,⁎⁎⁎ 15.2±0.66c,⁎⁎⁎

CAT (μmol/min/mg protein) 136.85±5.842 140.25±6.998 83.85±3.193a,⁎⁎⁎,b,⁎⁎⁎ 134.63±5.732b,⁎,c,⁎⁎⁎ 125.97±5.298a,⁎,b,⁎,c,⁎⁎⁎

GST (μmol/min/mg protein) 3.66±0.173 3.41±0.169 1.91±0.085a,⁎⁎,b,⁎⁎ 3.74±0.176c,⁎⁎ 3.69±0.174c,⁎⁎

GR (nmol/min/mg protein) 6.014±0.299 6.635±0.232 1.792±0.079a,⁎⁎⁎,b,⁎⁎⁎ 7.835±0.292a,⁎⁎,b,⁎,c,⁎⁎⁎ 7.659±0.283a,⁎,b,⁎,c,⁎⁎⁎

GPx (nmol/min/mg protein) 119.94±4.79 122.14±4.99 78.41±2.92a,⁎⁎⁎,b,⁎⁎⁎ 112.43±4.64c,⁎⁎⁎ 109.20±4.29b,⁎,c,⁎⁎⁎

Values are expressed as mean±SD, for 6 animals in each group.a Indicates that it differs significantly from normal.b Indicates that it differs significantly from TAU.c Indicates that it differs significantly from ALX.

⁎ Pb0.05.⁎⁎ Pb0.01.⁎⁎⁎ Pb0.001.

302 J. Das et al. / Toxicology and Applied Pharmacology 258 (2012) 296–308

cholesterol, triglycerides and LDL levels in diabetic rats which canlead to increased atherosclerosis and incidence of heart disease(Stamler et al., 1993). Taurine, on the other hand, improved theconditions of plasma cholesterol, triglycerides, LDL as well as themarkers of atherogenic risk, total cholesterol/HDL and LDL/HDLratios in ALX-induced diabetic animals indicating its lipid loweringactivities via unique anti-atherogenic properties. Taurine reducedthe triglyceride and total cholesterol levels in diabetic rats due to7α-hydroxylase activation that participates in the synthesis ofbiliary acids (Nakaya et al., 2000; Yokogoshi et al., 1999). Sincetaurine increased the insulin level, it would increase the level ofLDL receptor and as a result LDL-cholesterol level was decreased(Suryawanshi et al., 2006).

High levels of circulating cardiac dysfunction/damage markerssuch as, LDH and uric acid represent a sensitive predictor of increasedcardiac complications (Howard-Alpe et al., 2006). Owing to thedisturbances in the cardiac cell membrane integrity, LDH enters intothe blood (Zhou et al., 2008). Diabetic patients with high serum uricacid levels are associated with increased risk of fatal and nonfatalstroke (Lehto et al., 1998). The significant elevation observed in thelevel of serum uric acid in the ALX-induced diabetic rats could bedue to the increased abundance and activity of xanthine oxidase,(Ekelund et al., 1999). In our study, taurine showed a significantdecrease in the elevated levels of these circulating cardiac damagemarkers, thereby proving it to be an effective cardioprotectant underdiabetic conditions.

The toxic effect of ALX in the pancreas is preceded by its rapiduptake by the β cells and generation of ROS. ALX in the presence ofintracellular thiols (reduced glutathione, cysteine and protein-boundsulfhydryl groups), ascorbates or glucokinase undergoes reduction,forming dialuric acid. It is then re-oxidized to ALX via formation ofsuperoxide radicals and establishes a redox cycle. These radicals further

Table 5Effect of alloxan and taurine on the serum lipid profile.

Parameters Normal control TAU treated A

Total cholesterol (mg/dL) 54.55±1.728 53.58±1.679 1HDL cholesterol (mg/dL) 20.24±0.088 18.32±0.816LDL cholesterol (mg/dL) 21.81±1.0805 14.16±0.608

Total cholesterol/HDL 2.50±0.115 2.92±0.126LDL/HDL 1.08±0.044 0.77±0.028a,⁎

Triglyceride (mg/dL) 107.83±4.391 105.54±4.277 2

Values are expressed as mean±SD, for 6 animals in each group.a Indicates that it differs significantly from normal.b Indicates that it differs significantly from TAU.c Indicates that it differs significantly from ALX.

⁎ Pb0.05.⁎⁎ Pb0.01.⁎⁎⁎ Pb0.001.

produce hydrogen peroxide by dismutation reaction. In the presence ofhydrogen peroxide and Fe2+, highly reactive hydroxyl radicals (OH ˙)are formed (Szkudelski, 2001). The elevated levels of (OH ˙) havealso been reported in plasma and other tissues of diabetic animals(Winiarska et al., 2009). The formation and accumulation of ad-vanced glycation end products (AGEs) in various diabetic tissuesare also associated with altered protein (including antioxidantenzymes) structure and function and are able to activate cellularresponses that generate ROS (Giardino et al., 1998; Yeh et al.,2001). ROS can also be formed via glucose autooxidation (Wolffand Dean, 1987). In our present study, we have shown that ALX-induced diabetic condition resulted in an increase in the lipid perox-idation and protein carbonylation in cardiac tissue. Enhanced lipidperoxide may be due to the increased glycation of proteins, glucoseautooxidation, decreased SOD enzyme activity and lack of GSH(Suryawanshi et al., 2006). In addition, ALX also increased xanthineoxidase activity and decreased the activities of antioxidant enzymes(SOD, CAT, GST, GR, and GPx) and the redox ratio (GSH/GSSG). Allthese are direct indicators of oxidative stress. Xanthine oxidase isresponsible for the formation of uric acid and also serves as animportant biological source of ROS (superoxide) that contribute tooxidative damage involved in many pathological processes such asdiabetes, atherosclerosis, cancer and aging (Akhileshwar et al.,2007; Cos et al., 1998; Thomson et al., 2007). However, taurine treat-ment in the ALX-diabetic rats reduced lipid peroxidation, proteincarbonylation, and xanthine oxidase activity and restored the activ-ities of antioxidant enzymes. Therefore, the increased activities ofSOD, CAT, GR and GPx due to taurine treatment may help to avoidthe deleterious effects of the free radicals generated during diabetes.In the hearts of diabetic rats taurine-evoked increase in GSH/GSSGratio is achieved via the normalization of GSSG content, whichseems to result from the elevated GR activity. This beneficial effect

LX treated ALX&TAU ALX+TAU

00.48±4.024a,⁎⁎⁎,b,⁎⁎⁎,⁎⁎ 56.46±1.823c,⁎⁎⁎ 61.24±2.062c,⁎⁎⁎

17.80±0.69 21.98±0.075 20.24±0.06948.83±1.442a,⁎⁎⁎,b,⁎⁎⁎ 22.71±0.936b,⁎,c,⁎⁎⁎ 22.72±0.936b,⁎,c,⁎⁎⁎

5.64±0.182a,⁎⁎⁎,b,⁎⁎⁎ 2.57±0.119c,⁎⁎⁎ 3.03±0.102c,⁎⁎⁎

2.74±0.117a,⁎⁎⁎,b,⁎⁎⁎ 1.03±0.042b,⁎,c,⁎⁎⁎ 1.12±0.046a,⁎,c,⁎⁎⁎

16.44±9.822a,⁎⁎⁎,b,⁎⁎⁎ 121.22±5.061b,⁎,c,⁎⁎⁎ 114.34±4.717c,⁎⁎⁎

Fig. 5. Hematoxylin and eosin stained cardiac section of normal, ALX and taurine-treated rats (10×). Arrows indicate disorganization of normal radiating pattern ofcell plates in the heart in ALX-exposed animals.

A) NFkB

tubulin

Nor Tau Alx Alx& Alx+Tau Tau

C

i

0

200

400

600

ab**,c***ab**,c***

ab***

TAU

ALX&TAU

ALX+TAUALX

TN

Fa

(pg/

mg

prot

ein)

NOR

65kD

48kD

γ

Fig. 6. Effect of taurine and diabetes on NFκB nuclear translocation (A), myeloperoxidase actiData represent the average±SD of 6 separate experiments in each group. “a” indicates diffediffers significantly from ALX. ⁎⁎Pb0.01, and ⁎⁎⁎Pb0.001.

303J. Das et al. / Toxicology and Applied Pharmacology 258 (2012) 296–308

of taurine could be directly related to its antioxidative nature whichis in agreement with the previous findings (Das et al., 2010a; Ghoshet al., 2009).

Diabetes is also known as an inflammation-prone disease apartfrom the altered metabolic condition. Hyperglycemia-induced ROSproduction stimulates the signal transduction pathway for the activa-tion of the transcription factor NF-κB which helps to control theexpression of numerous genes activated during inflammation (i.e.,cytokines, chemokines, growth factors, immune receptors, cellularligands, and adhesion molecules) (Kuhad and Chopra, 2009). Thisleads to cardiac dysfunction and exacerbates the severity of diabetes(Sun et al., 2011). In our present study, augmented cardiac inflamma-tion was observed as evidenced by increased NFκB nuclear transloca-tion and increased TNF-α and IL-6 levels. Cardiac inflammation wasfurther confirmed by increased myeloperoxidase (MPO) activity.MPO, a major neutrophil protein, is also present in monocytes. Itproduces hypochlorous acid (HOCl) and other reactive oxidants byusing superoxide and hydrogen peroxide generated by the neutro-phil oxidative burst. HOCl is a potent mediator of inflammation(Winterbourn et al., 2000). However, being an antioxidant, taurinecould effectively antagonize the toxic effect of HOCl by scavengingand forming a stable product, taurine-chloramine which possessesanti-inflammatory and cytoprotective properties (Schuller-Levisand Park, 2003). Besides the inhibition of MPO activity, taurine alsodecreased the levels of proinflammatory cytokines (TNF-α and

B

D

i

0

20

40

60

80

ab**,c***ab**,c***

ab***

IL-6

(pg/

mg

tiss

ue)

TAU

ALX&TAU

ALX+TAUALX

NOR

0

5

10

15

20

ab**,c***ab**,c***

ab***

TAU

ALX&TAU

ALX+TAUALX

Myl

eope

roxi

dase

act

ivit

y(U

/mg

prot

ein)

NOR

vity (B) and proinflammatory cytokines; TNF-α (C), IL-6 (D) after 3 weeks of treatment.rs significantly from normal; “b” indicates differs significantly from TAU; “c” indicates

A) phospho IR

Total IR

B) phospho IRS1

Total IRS1

C) phospho Akt

Total Akt

actin

D) Glut4

Nor Tau Alx Alx& Alx+Tau Tau

95kD

110kD

132kD

165kD

56kD

56kD

42kD

54kD

i

0.0

0.3

0.6

0.9

1.2

1.5

b**,c*b*,c**,d*

ab**

TAU

ALX&TAU

ALX+TAU

ALX

Pho

spho

/tot

al I

R(a

rbit

rary

uni

t)

NOR

i

0.0

0.3

0.6

0.9

1.2

1.5

b**,c*b*,c**,d*

ab**

TAU

ALX&TAU

ALX+TAUALX

Pho

spho

/tot

al I

RS1

(arb

itra

ry u

nit)

NOR

i

0.0

0.3

0.6

0.9

1.2

1.5

c*c**,d*

ab**

TAU

ALX&TAU

ALX+TAUALX

Pho

spho

/tot

al A

kt(a

rbit

rary

uni

t)

NOR

i

0.0

0.3

0.6

0.9

1.2

1.5

c*c**,d*

ab**

TAU

ALX&TAU

ALX+TAUALX

GL

UT

4(a

rbit

rary

uni

t)

NOR

β

Fig. 7. Effect of taurine and diabetes on insulin-mediated glucose transport signaling pathways. Panel A: phospho (tyrosine) and total IR, panel B: phospho (tyrosine) and total IRS1,panel C: phospho (serine) and total Akt, and panel D: membrane translocation of GLUT4. Data represent the average±SD of 6 separate experiments in each group. “a” indicates thatit differs significantly from normal; “b” indicates that it differs significantly from TAU; “c” indicates that it differs significantly from ALX; “d” indicates that it differs significantly fromALX&TAU. ⁎Pb0.05, ⁎⁎Pb0.01.

304 J. Das et al. / Toxicology and Applied Pharmacology 258 (2012) 296–308

IL-6) and NFκB nuclear translocation. The inhibition of NFκB nucleartranslocation by taurine has also been reported by other investigators(Giri et al., 2000 and Gurujeyalashmi et al., 2000).

Several studies including our own have revealed that taurine isinvolved in glucose homeostasis, although the specific molecularmechanisms are still unknown. On the other hand, numerous studieshave also reported that taurine treatment did not reduce plasma

glucose level in diabetic rats (Li et al., 2005; Obrosova et al., 2001).However, this inconsistencymight result due tomany factors, includingthe route of taurine administration. It is obvious that, when taurine issupplemented through drinking water, it could be better absorbedand in consequence, more effective than its administration throughfood stuffs (Winiarska et al., 2009). Several investigators have reportedthat taurine treatment before diabetic onset suppressed hyperglycemia

A) Bad E

B) Bcl2

C) Bax

D) BclxL

F) Cyt c (cytosolic) G) Cyt c (mitochondrial)

Nor Tau Alx Alx& Alx+

H) caspase 9 Tau Tau

I) caspase 3

actin

Nor Tau Alx Alx& Alx+Tau Tau

J1 2 3 4 5 6

17kD

15kD

35kD

26kD

25kD

23kD

i

0.0

1.5

3.0

4.5

6.0

ab**,c***ab**,c***

ab***

TAU

ALX&TAU

ALX+TAU

ALX

Bad

/Bcl

-2(a

rbit

rary

uni

t)

NOR

i

0

2

4

6

8

ab**,c***ab**,c***

ab***

TAU

ALX&TAU

ALX+TAUALX

Bax

/Bcl

xL(a

rbit

rary

uni

t)

NOR

30kD

42kD

i

0

2

4

c***c***

ab***

TAU

ALX&TAU

ALX+TAU

ALX

Cas

pase

3(a

rbit

rary

uni

t)

NOR

β

Fig. 8. Effect of taurine and diabetes on cardiac apoptosis. Panel A: Bad, panel B: Bcl2, panel C: Bax, panel D: BclxL, panel E: mitochondrial membrane potential, panel F: cytosoliccytochrome c, panel G: mitochondrial cytochrome c, panel H: caspase 9, panel I: caspase 3, and panel J: DNA fragmentation pattern on agarose/EtBr gel. Lane 1: Marker (1 kb DNAladder); lane 2, lane 3, lane 4, lane 5, and lane 6: DNA isolated from normal, ALX intoxicated, taurine simultaneous, post treated and taurine only treated rats respectively. Arrowsindicate ladder formation. Data represent the average±SD of 6 separate experiments in each group. “a” indicates that it differs significantly from normal; “b” indicates that it differssignificantly from TAU; “c” indicates that it differs significantly from ALX. ⁎⁎Pb0.01, and ⁎⁎⁎Pb0.001.

305J. Das et al. / Toxicology and Applied Pharmacology 258 (2012) 296–308

in streptozotocin-induced type 1 diabetic rats (Alvarado-Vasquez et al.,2003; Tokunaga et al., 1983). On the other hand, it has also beenreported that treatment with taurine after diabetic onset failed to sup-press hyperglycemia in type 1 diabetic rats, but found to be effective iftreated for longer time periods (Di Leo et al., 2004; Vasqueza et al.,2003; Yao et al., 2009). Therefore, the treatment time and routes ofadministration of taurine are important for its beneficial action intreating diabetes. Several hypotheses concerning the hypoglycemiceffect of taurine are: protection of β-cells/β-cell insulin secretion

(Chang and Kwon, 2000; Cherif et al., 1998; Gavrovskaya et al.,2008; Kaplan et al., 2004); enhanced insulin sensitivity (Carneiroaet al., 2009; Colivicchi et al., 2004; Wu et al., 2010), insulin like effect(Kulakowski and Maturo, 1984; Maturo and Kulakowski, 1988), di-minished glucose absorption from gastrointestinal tract (Kim et al.,2006) and its accelerated utilization by peripheral tissues(Nandhini et al., 2004) although there is no earlier report concerningthe insulin sensitivity or insulin mimetic effect of taurine in ALX-induced type I diabetic rats. Like other tissues, insulin signaling via

Fig. 9. TUNEL staining in cardiac tissue sections in experimental animals. Normal: pancreatic section of normal rat pancreas (×10); ALX: pancreatic section from the diabetic group(×10); ALX&TAU: pancreatic section from the taurine simultaneous and ALX+TAU: taurine post treated rats respectively (×10). Arrows indicate TUNEL positive nucleus.

306 J. Das et al. / Toxicology and Applied Pharmacology 258 (2012) 296–308

Akt/GLUT4 pathways plays a key role in cardiac glucose uptake(Brownsey et al., 1997). Cardiac tissues obtain its energy by theoxidation of fatty acids, glucose, lactate and ketone bodies(Rodrigues andMcNeill, 1992). During diabetes or insulin resistance,the energy metabolism is altered in heart due to less glucose uptake;as a result glucose utilization is decreased and fatty acid consump-tion is increased. This disturbance of glucose metabolism worsenmetabolic efficiency of cardiomyocytes and plays an important rolein the development of atherosclerotic heart disease, left ventricularhypertrophy and dysfunction, heart failure and cardiomyopathy(Murakami et al., 2004). In our present study, we observed thatunder diabetic conditions, phosphorylation at tyrosine residue ofIR and IRS1 proteins; and phosphorylation at serine residue of Aktprotein were decreased. We also observed decreased GLUT4

Fig. 10. Schematic diagram of the ALX induced dia

expressions in diabetic hearts. However, administration of taurineto diabetic rats significantly improved this insulin signaling path-way and restored GLUT4 translocation to the plasma membranesfor the uptake of glucose. These results are also supported by previ-ous findings that taurine may act directly on the insulin receptor tofacilitate glucose uptake (Kulakowski and Maturo, 1984; Maturo andKulakowski, 1988) and suggest that taurine seems to be beneficialto decrease diabetes-associated cardiac complications via increasedglucose uptake in the heart.

In the present study, we have also investigated the involvementof ALX-mediated intrinsic apoptotic cell death pathway in cardiactissues. Immunoblot analyses revealed that taurine treatment pro-tected ALX-induced apoptotic cell death in cardiac tissues via theregulation of pro-apoptotic proteins, such as Bax/Bad and anti-

betes and its prevention/curation by taurine.

307J. Das et al. / Toxicology and Applied Pharmacology 258 (2012) 296–308

apoptotic proteins, such as Bcl-2/Bcl-xL, lowering of mitochondrialmembrane potential, enhancing the release of cytochrome c intocytosol and the cleavage of caspase-9/3. In addition, ALX-inducedcardiac apoptotic cell death was also evident from DNA fragmen-tation and TUNEL assays. However, taurine treatment protectedcardiac tissue from ALX-induced DNA breakdown and decreasedthe number of TUNEL positive nucleus.

In conclusion, the results of the present study demonstrates that(i) taurine administration exerts anti-hyperglycemic effects, controlsblood glucose levels and restores body weight in diabetic animals; (ii)taurine increases the level of insulin in diabetic rats (this effect ismore pronounced in taurine simultaneous treated group comparedto taurine post treatment); (iii) taurine restores GLUT4 expressionin the plasma membranes of the heart tissues via insulin dependentmechanism (this effect is more pronounced in taurine post treatedgroup compared to taurine simultaneous treatment group), therebyinducing glucose uptake in diabetic conditions; (iv) taurine exertsantioxidant, antihyperlipidemic and antiinflammatory activities and(v) taurine protects cardiac tissue against apoptosis induced by ALX(as indicated in the proposed scheme of Fig. 10). Since taurine is anendogenous substance in the body, it is not associated with toxicityor drug dependence, therefore well tolerated when used clinically(Chang, et al., 2004). Taken together, our results suggest that taurinesupplementation may be considered as an applicable approach inpreventing or treating diabetes and associated cardiac complications.

Conflict of interestThe authors have declared that no conflict of interest exists.

Acknowledgments

The authors are grateful to Mr. Prasanta Pal for excellent technicalassistance for the study. The financial support from Bose Institute hasalso been acknowledged.

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